Dermatophagoides farinae allergen-specific ige antibody detection kit and application thereof

Abstract

This invention relates to the field of biodetection technology, and more particularly to a detection kit for house dust mite allergen-specific IgE antibody and its application. This invention prepares a house dust mite allergen supernatant solution by pretreating and biotinylating house dust mite extract. Combined with a magnetic microparticle suspension and a mouse anti-human IgE antibody solution, it enables rapid, simple, highly sensitive, highly specific, fully automated, and fully quantitative detection of small amounts of serum / plasma samples. The detection time is short, the detection cost is low, and it can be widely applied.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a detection kit for house dust mite allergen-specific IgE antibody and its application. Background Technology

[0002] Allergic reactions are overreactions of the human immune system to repeated exposure to harmless substances in the environment. These substances, commonly known as allergens, are the main culprits behind allergic diseases. The mechanism is as follows: after an allergen enters the body, it induces B cells to produce specific IgE (sIgE). sIgE binds to receptors on the surface of mast cells or basophils, sensitizing the body. When the same allergen re-enters the sensitized body, it binds to sIgE antibodies, triggering a series of biochemical reactions in the cell membrane, releasing many active mediators such as histamine, proteolytic enzymes, heparin, and chemokines; thus producing an allergic reaction. This underscores the importance of allergen detection.

[0003] House dust mite allergens are a significant inhaled allergen and a major factor contributing to indoor allergic diseases. Studies have shown that 60% of adult asthma patients are allergic to house dust mites, and this figure is even higher in children, reaching 80%. In vitro quantitative detection of house dust mite allergen-specific IgE antibodies in human serum or plasma can aid in the diagnosis of house dust mite allergy. Simultaneously, quantitative detection of sIgE can assess disease risk, enabling early diagnosis and risk avoidance, and providing medication guidance for desensitization therapy.

[0004] Currently, in vitro diagnostic reagents for the quantitative detection of house dust mite allergen-specific IgE antibodies in human serum or plasma are available from one imported product and four domestic products in China. All of these reagents operate on the immunological principle of antigen-antibody reaction. The benchmark product, the house dust mite d1 allergen-specific IgE antibody detection kit (fluorescent immunoassay), utilizes an allergen covalently coated on ImmunoCAP to react with specific IgE in the patient's sample. Enzyme catalysis enhances the chemiluminescent substrate, producing fluorescence; higher fluorescence intensity indicates a higher concentration of specific IgE in the patient's serum. However, this kit is expensive and time-consuming (approximately 2 hours). Therefore, developing a kit with high specificity, high sensitivity, low cost, and short processing time is of great significance for the clinical detection of house dust mites. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a detection kit for house dust mite allergen-specific IgE antibodies and its application. The present invention provides a kit for detecting house dust mite allergen-specific IgE antibodies using magnetic microparticle chemiluminescence immunoassay after pretreatment and biotinylation modification of house dust mite extract, and the application of this kit in clinical testing.

[0006] The present invention provides a reagent combination comprising a magnetic microparticle suspension, a house dust mite allergen solution and / or a mouse anti-human IgE antibody solution; wherein the house dust mite allergen solution is a supernatant that has been biochemically modified and diluted after centrifugation pretreatment.

[0007] This invention, after centrifuging a solution of house dust mite allergens, revealed that the precipitate lacked the major allergenic proteins Derp1 and Derp2, while the supernatant contained higher concentrations and purity of these two proteins. This indicates that centrifugation of the house dust mite allergen solution effectively removes impurities and interfering substances from the original extract, reduces protein background, and purifies the major allergenic proteins. Furthermore, this pretreatment step is simple and time-efficient.

[0008] In the reagent combination described in this invention, the centrifugal pretreatment step of the house dust mite allergen solution includes: diluting the house dust mite allergen extract with deionized water to 2-5 mg / mL, allowing it to stand for 2-5 hours, and then centrifuging at 80,000-13,000 g for 3-10 minutes. Preferably, the house dust mite allergen extract is diluted with deionized water to 0.2-2 mg / mL.

[0009] Furthermore, the house dust mite allergen solution is modified with biotin; the modification steps include: mixing the house dust mite allergen solution with a biotinylation reagent, shaking the mixture at 2-8°C for 0.5-3 hours, removing the free biotin, and obtaining the biotinylated house dust mite allergen.

[0010] The molar ratio of the house dust mite allergen to the biotinylation reagent is 1:5 to 1:20, preferably 1:10; wherein the buffer solution used for dilution after biotinylation modification is a 0.05M Tris-NaCl buffer containing 1wt% to 5wt% bovine serum albumin, 0.1wt% to 0.5wt% preservative, and 0.1wt% to 0.5wt% Triton 100, with a pH of 7 to 8. The preservative is Proclin 300.

[0011] In the reagent combination described in this invention, the magnetic bead content in the magnetic microparticle suspension is 0.5–3 mg / mL, preferably 1–2 mg / mL; the magnetic microparticles in the magnetic microparticle suspension are coated with streptavidin; the step of coating the magnetic microparticles with streptavidin includes washing the magnetic microparticles four times with 0.05 M tris(hydroxymethyl)aminomethane hydrochloride and then resuspending them in a tris(hydroxymethyl)aminomethane-sodium chloride buffer containing human serum albumin; the combination of these buffers plays a good role in improving the sensitivity and accuracy of the reagent kit detection, and is superior to the effect of other buffers.

[0012] The concentration of the mouse anti-human IgE antibody solution is 0.1–1.5 μg / mL, preferably 1.0 μg / mL; the mouse anti-human IgE antibody is labeled with horseradish peroxidase.

[0013] This invention involves biotinylation labeling of the supernatant, precipitate, and mixture of natural house dust mite extracts. Testing of 160 samples revealed that the detection results from the supernatant of the natural house dust mite extract were more accurate, and the positive concordance rate with the ImmunoCAP kit (the "gold standard" for allergen diagnosis) was higher than that from the precipitate and mixture of the natural house dust mite extract. This indicates that using the supernatant of the natural house dust mite extract as an allergen for detection can improve the sensitivity of the kit.

[0014] This invention provides the application of the reagent combination in a kit for preparing a specific IgE antibody for detecting house dust mite allergens.

[0015] The present invention also provides a quantitative detection kit for house dust mite allergen-specific IgE antibodies, which includes the reagent combination described in the present invention and acceptable adjuvants.

[0016] Furthermore, the kit described in this invention also includes luminol reagent, peroxide reagent and / or calibrators.

[0017] The present invention also provides a method for detecting IgE antibodies specific to house dust mite allergens, which includes detecting a sample using the reagent combination or the kit described in the present invention; the sample is the supernatant after centrifugation pretreatment.

[0018] Specifically, in embodiments of the present invention, the detection method includes the following steps:

[0019] 1) Add 20 μL of calibrator or test sample to the reaction vessel;

[0020] 2) Add 20 μL of streptavidin-coated magnetic microparticle suspension and 20 μL of biotinylated house dust mite allergen solution to the reaction vessel and incubate at 37±0.5℃ for 15 min;

[0021] 2) After incubation, the supernatant is removed by magnetic separation, and the mixture is washed three times with washing solution;

[0022] 3) Add 60 μL of horseradish peroxidase-labeled mouse anti-human IgE antibody solution and incubate at 37±0.5℃ for 15 min;

[0023] 4) After incubation, magnetic separation is performed to remove the supernatant, followed by washing with washing solution three times;

[0024] 5) Finally, add 50 μL of substrate A solution (containing luminol reagent) and 50 μL of substrate B solution (containing peroxide reagent), shake to mix, and measure the chemiluminescence intensity (RLU);

[0025] 6) Select an appropriate curve fitting method to calculate the concentration (IU / mL) of house dust mite allergen-specific IgE in the sample.

[0026] The methods provided by this invention can be for diagnostic purposes or non-diagnostic purposes. For example, they include diagnostic testing methods for human or animal bodies, or ex vivo samples derived from human or animal bodies; they also include scientific or other non-diagnostic testing methods for environmental or simulated samples.

[0027] The present invention provides a method for obtaining extract supernatant by centrifugation pretreatment of natural antigen extracts, followed by biotinylation modification using the extract supernatant. This method improves antigen loading and purity, removes impurities and interfering substances from the original extract, and effectively increases the positive detection rate and sensitivity of the kit. The kit provided by the present invention is suitable for magnetic particle chemiluminescence instrument platforms and has the advantages of fully automated detection, short detection time, and low detection cost. Attached Figure Description

[0028] Figure 1 This study investigated the effect of centrifugation pretreatment on the content of allergenic proteins in crude extracts of house dust mite allergens. Detailed Implementation

[0029] This invention provides a house dust mite allergen-specific IgE antibody detection kit and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0030] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0031] The present invention will be further illustrated below with reference to the embodiments:

[0032] Example 1: Pretreatment of House Dust Mite Allergen Solution

[0033] The lyophilized powder of natural extract of house dust mites (purchased from Zhengzhou Yimeno Biotechnology Co., Ltd.) was diluted with purified aqueous solution and equilibrated at room temperature for 2-5 hours. Then, it was centrifuged at high speed (80,000-13,000 g) for 3-10 minutes. The supernatant, precipitate, and uncentrifuged mixture of the extract were collected and subjected to SDS-PAGE electrophoresis to determine protein content (performed by Zhengzhou Yimeno). The results are as follows: Figure 1 As shown.

[0034] SDS-PAGE protein electrophoresis results showed that after dilution and centrifugation pretreatment of the natural allergen extract of house dust mites, the extract precipitate lacked the main allergenic proteins Der p1 and Der p2 of house dust mites; compared with the extract mixture, the protein bands in the extract supernatant were more obvious and clear and the background was cleaner.

[0035] Example 2: Reagent Preparation in the Kit

[0036] 1. The preparation steps of biotinylated house dust mite allergen extract solution are as follows:

[0037] (1) The molar ratio of house dust mite allergen to biotinylation reagent was 1:10; the concentration of house dust mite allergen labeling was 1 mg / mL. The biotinylation reagent was Sulfo-NHS-LC-biotin reagent from Thermo Scientific.

[0038] (2) After shaking the reaction at 2-8℃ for 0.5-3 hours, unreacted biotin is removed using a biotin removal column, and the resulting liquid is biotinylated house dust mite allergen extract.

[0039] (3) Biotinylated house dust mite allergen extract was prepared into a 10ug / mL solution using 0.05M Tris-NaCl buffer (pH=7-8) containing 1%-5% bovine serum albumin (BSA), 0.1%-0.5% preservative (Proclin 300), and 0.1%-0.5% polyethylene glycol monooctylphenyl ether (Triton 100).

[0040] 2. The preparation steps of the streptavidin-coated magnetic microparticle suspension are as follows:

[0041] (1) Resuspend the magnetic microparticle stock solution in 0.05M Tris-HCl buffer (pH=7-8), wash repeatedly 4 times (5-10 min / time), and perform magnetic separation.

[0042] (2) The magnetic microparticles were resuspended in a 0.05M Tris-NaCl buffer (pH=7-8) containing 1-5% human serum albumin (HSA) to obtain a streptavidin-coated magnetic microparticle suspension with a concentration of 1-2 mg / mL.

[0043] 3. The preparation steps of horseradish peroxidase-labeled mouse anti-human IgE antibody solution are as follows:

[0044] HRP-labeled mouse anti-human IgE antibody was prepared into a 0.1-1.5 μg / mL solution using 0.05 M Tris-NaCl buffer (pH = 7-8) containing 1-5% BSA, 0.1-0.5% casein, 0.1-0.5% Proclin 300, and 0.1-0.5% Triton 100.

[0045] Example 3: Detection of supernatant, precipitate, and mixture of house dust mite allergen extracts.

[0046] Three biotinylated house dust mite allergen extract solutions were obtained by biotinylating the supernatant, precipitate, and mixture of extracts. The kit described in this invention and the ImmunoCAP kit (the gold standard for allergen diagnosis) were used to detect 160 samples in parallel. The analysis was performed using the Antu Bio A2000Plus fully automated chemiluminescence analyzer. The steps are as follows:

[0047] 1) First, add the calibrator or test sample from the 20uL specific IgE antibody detection kit (magnetic microparticle chemiluminescence method) to the reaction vessel.

[0048] 2) Add 20 μL of streptavidin-coated magnetic microparticle suspension and 20 μL of biotinylated house dust mite allergen solution to the reaction vessel and incubate at 37±0.5℃ for 15 min;

[0049] 2) After incubation, the supernatant is removed by magnetic separation, and the mixture is washed three times with washing solution;

[0050] 3) Then add 60uL of horseradish peroxidase-labeled mouse anti-human IgE antibody solution and incubate at 37±0.5℃ for 15min;

[0051] 4) After incubation, magnetic separation is performed to remove the supernatant, followed by washing with washing solution three times;

[0052] 5) Finally, add 50 μL of substrate A solution (containing luminol reagent) and 50 μL of substrate B solution (containing peroxide reagent), shake to mix, and measure the chemiluminescence intensity (RLU);

[0053] 6) Select an appropriate curve fitting method. This kit recommends using a five-parameter (lin / log) fitting standard curve. The concentration of the calibrator in the specific IgE antibody detection kit (magnetic particle chemiluminescence method) is used as the x-axis, and the corresponding chemiluminescence signal value is used as the y-axis to establish a calibration curve. The concentration value (IU / mL) of house dust mite allergen-specific IgE in the test sample is calculated based on the chemiluminescence signal value of the test sample.

[0054] The test results are shown in the table below:

[0055] Table 1

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] The results showed that the centrifugation pretreatment step of the natural extract of house dust mites, obtaining the supernatant of the natural extract as the house dust mite allergen, and then performing biotinylation modification can improve the positive detection rate (sensitivity) of the kit.

[0063] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reagent combination, characterized in that It includes a magnetic microparticle suspension, a house dust mite allergen solution, and a mouse anti-human IgE antibody solution; wherein the house dust mite allergen solution is a supernatant that has been biochemically modified and diluted after centrifugation pretreatment; The biochemical modification step includes: mixing a house dust mite allergen solution with a biotinylation reagent, shaking the mixture at 2-8°C for 0.5-3 hours, removing free biotin, and obtaining biotinylated house dust mite allergen. The molar ratio of the house dust mite allergen to the biotinylation reagent is 1:5 to 1:

20. The buffer solution used for dilution after biotinylation modification is a 0.05 M Tris-NaCl buffer containing 1 wt% to 5 wt% bovine serum albumin, 0.1 wt% to 0.5 wt% preservative, and 0.1 wt% to 0.5 wt% Triton 100, with a pH of 7 to 8; wherein the preservative is Proclin 300. The magnetic microparticle suspension contains 0.5~3 mg / mL of magnetic microparticles; the magnetic microparticles in the magnetic microparticle suspension are coated with streptavidin. The concentration of the mouse anti-human IgE antibody solution is 0.1~1.5 μg / mL; the mouse anti-human IgE antibody is labeled with horseradish peroxidase.

2. The combination of agents according to claim 1, characterized in that, The steps of centrifugation pretreatment of the house dust mite allergen solution include: diluting the house dust mite allergen extract with deionized water to 2-5 mg / mL, letting it stand for 2-5 h, and then centrifuging at 80000-13000 g for 3-10 min.

3. The use of the reagent combination of claim 1 or 2 in a kit for preparing a specific IgE antibody for detecting house dust mite allergens.

4. A quantitative detection kit for house dust mite allergen-specific IgE antibody, characterized by, It includes the reagent combination as described in claim 1 or 2, as well as acceptable adjuvants.

5. The kit of claim 4, wherein It also includes luminol reagent and / or peroxide reagent.

6. A method for detecting house dust mite allergen-specific IgE antibodies for non-diagnostic purposes, characterized in that, This includes detecting a sample using the reagent combination of claim 1 or 2 or the kit of claim 4 or 5; the sample is the supernatant after centrifugation pretreatment.

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