Mushroom allergen IgE antibody detection kit
By pretreatment and photobiotin coupling of mushroom allergens, the problems of low sensitivity and cross-reaction of mushroom allergen IgE antibody detection kits in the prior art are solved, and higher detection sensitivity and accuracy are achieved.
Patent Information
- Application Number
- CN202411658920.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-03-07
Smart Images
Figure CN119574882A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of in vitro diagnostic kits, and specifically relates to a mushroom allergen IgE antibody detection kit. Background Art
[0002] Some people will experience allergies after eating mushrooms, and the symptoms mainly include skin symptoms, respiratory symptoms and gastrointestinal symptoms. However, when the above symptoms appear, it cannot be directly determined as a mushroom allergy, and targeted testing is required. Mushroom-specific IgE antibodies are produced in the serum of people with mushroom allergies. The detection of mushroom-specific IgE antibodies by magnetic particle chemiluminescence can indirectly determine whether it is a mushroom allergy.
[0003] The principle of magnetic particle chemiluminescence detection of mushroom allergen IgE antibodies is that biotin-labeled mushroom allergens bind to mushroom-specific IgE antibodies in serum and are fixed to streptavidin-labeled nanomagnetic particles. After washing, alkaline phosphatase-labeled anti-human IgE secondary antibodies are added to form a solid-phase antigen-antibody-enzyme-labeled secondary antibody complex. Unbound enzyme-labeled antibodies and other substances are removed through washing, and then luminescent substrates are added. The enzyme on the enzyme-labeled antibody catalyzes the luminescent substrate to emit photons. The luminescence intensity is proportional to the specific IgE content in the sample. The content of specific IgE antibodies in the sample can be obtained by comparing the luminescence intensity with the working curve.
[0004] The existing mushroom allergen IgE antibody detection kit has a low sensitivity, resulting in clinical missed detections. At the same time, in clinical use, a small number of samples have been found to have cross-reactions with mold allergens, resulting in deviations in diagnostic results. Summary of the invention
[0005] The technical problem solved by the present invention is to overcome the deficiencies of the prior art and provide a mushroom allergen IgE antibody detection kit with higher sensitivity and more accurate detection results.
[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0007] A mushroom allergen IgE antibody detection kit comprises biotinylated mushroom allergens, streptavidin-labeled nanomagnetic particles, alkaline phosphatase-labeled anti-human IgE secondary antibodies, sample diluents and luminescent substrates, wherein the biotinylated mushroom allergens are mushroom allergens that are labeled with photosensitive biotin and have a molecular weight of less than 50 kDa and have been subjected to heat treatment.
[0008] In an embodiment of the present invention, the preparation method of the mushroom allergen is: using gel filtration chromatography to remove protein components with a molecular weight greater than or equal to 50 kDa from the mushroom allergen, and heating the remaining components in a water bath at above 80° C. to obtain the mushroom allergen.
[0009] Furthermore, the temperature of the water bath heating does not exceed 100°C.
[0010] Furthermore, the water bath heating time is 8 to 15 minutes.
[0011] Furthermore, the mushroom allergen IgE antibody detection kit also includes an allergen diluent, which is a MES buffer containing 0.8-1.0wt% NaCl, 0.8-1.5wt% BSA, 0.8-1.5wt% glycerol, 0.005-0.02wt% surfactant, and 0.05-0.2wt% preservative.
[0012] The surfactant is a conventional surfactant used in the art, preferably Triton X-100.
[0013] The preservative is a preservative commonly used in the art, preferably Proclin-300.
[0014] According to some preferred embodiments of the present invention, the photosensitive biotin is photosensitive biotin acetate.
[0015] The mushroom allergen before treatment used in the present invention is a commercially available mushroom allergen, and electrophoresis detection shows that it contains a large amount of components with a molecular weight greater than 50 kDa.
[0016] According to some preferred embodiments of the present invention, the mushroom allergen before treatment is a mushroom allergen with a product number of XPF150D3A2.5 from Stallergenes Greer.
[0017] In the embodiment of the present invention, the solid content of the streptavidin-labeled nanomagnetic particles is 0.1-1 mg / mL, and the particle size of the nanomagnetic particles is 0.5-1.5 μm.
[0018] According to some preferred embodiments of the present invention, the solid content of the streptavidin-labeled nanomagnetic particles is 0.6 mg / mL, and the particle size of the nanomagnetic particles is 0.904 μm.
[0019] In the embodiment of the present invention, the concentration of the alkaline phosphatase-labeled anti-human IgE secondary antibody is 0.1-1 μg / ml.
[0020] According to some preferred embodiments of the present invention, the luminescent substrate is AMPPD.
[0021] In the embodiment of the present invention, the luminescent substrate is a substrate solution for a fully automatic immunoassay system produced by Jiangsu Haooubo Biotechnology Co., Ltd. (article number: MY00073).
[0022] In the embodiment of the invention, the biotinylated mushroom allergen is preferably stored in a solid state after vacuum drying. In other embodiments, the biotinylated mushroom allergen can also be mixed with a sample diluent to prepare a mushroom allergen reagent with a concentration of 1 to 5 μg / mL for storage.
[0023] Compared with the prior art, the present invention has the following advantages:
[0024] The present invention pre-treats the existing mushroom allergens, including removing protein components with a molecular weight greater than or equal to 50 kDa, and heating the remaining components in a water bath at a temperature above 80°C, thereby improving the sensitivity of the mushroom allergen IgE antibody detection kit and avoiding misdiagnosis caused by cross-reactions between mushrooms and molds. At the same time, a photosensitive biotin coupling method is used to effectively solve the problem of low biotinylation efficiency of mushroom allergens, further improving the detection sensitivity, and thus greatly improving the accuracy of clinical detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The gel images obtained by electrophoresis of mushroom allergens before and after pretreatment;
[0026] Figure 2 The WES analysis results of mushroom allergens before and after pretreatment. DETAILED DESCRIPTION
[0027] In order to solve the problem of low sensitivity of existing mushroom allergen IgE antibody detection kits and the deviation between clinical test results and diagnostic results, the inventors of this application have conducted research on mushroom allergen IgE antibody detection kits. The inventors of this application believe that because the kit of this application is carried out on a magnetic particle chemiluminescence platform, the allergens are coupled to biotin and participate in the reaction, and the mushroom allergen raw materials themselves and the coupling efficiency of biotin will greatly affect the test results. However, there is no detailed record of the raw material components of mushroom allergens on the World Health Organization's website for allergen naming (https: / / www.allergen), so there is no standard for the raw materials for the development of this allergen, and it has always relied on imported allergens, such as the mushroom allergen with the product number XPF150D3A2.5 of Stallergenes Greer. During the development of the mushroom allergen IgE kit, it was found that the background of the material was very serious and the sensitivity was low, which could not meet the laboratory methodology comparison requirements. Therefore, the inventors of this application first optimized the allergens.
[0028] Studies have shown that clinical mushroom allergic reactions are mainly directed at protein components with smaller molecular weights, such as 8kDa, 9kDa, 10kDa, 11kDa and 15kDa. The SDS-PAGE electrophoresis analysis of purchased mushroom allergens showed that there were many small molecular weight protein bands and a very strong band at about 67kDa. Proteins less than 50kDa can cause allergic reactions in both raw and cooked mushrooms, while proteins around 67kDa can only cause allergic reactions in raw mushrooms and are heat-labile proteins. The high content of component proteins around 67kDa in the raw materials of mushroom allergens also relatively affects the relative content of small molecular weight proteins. Mushrooms themselves belong to large fungi. Proteins around 67kDa are easily cross-reacted with mycosporium by WES verification. If the allergic patient himself is positive for mycosporium antibodies, he will have allergic symptoms such as respiratory tract allergies similar to mold after eating raw mushrooms, which is easy to cause misdiagnosis. Therefore, the inventors of this application first used gel filtration chromatography to remove protein components ≥50kDa from the purchased mushroom allergen raw materials. Subsequent studies have found that adding heat treatment to destroy residual heat-labile protein allergenic components can effectively increase the positive compliance rate and reduce false positives from the perspective of the mushroom allergen raw materials themselves.
[0029] Subsequently, after optimizing the raw materials, the inventors of the present application focused on optimizing the biotin labeling of mushroom allergens. Ordinary biotin requires the selection of appropriate coupling agents during the coupling process, such as N-hydroxysuccinimide esters and thiodiformyl, etc. These coupling agents can react with functional groups such as amino or hydroxyl groups on the target molecule to form a covalent bond between biotin and the target molecule. However, photobiotin, under the irradiation of strong visible light, can generate highly reactive nitrenes, which are easy to combine with primary amino groups to form covalent bonds, which can reduce steric hindrance during coupling. This labeling scheme is simple and has high sensitivity. In the process of studying the biotinylation of mushroom allergens, it was found that the reactivity of the biotinylated mushroom allergen obtained after coupling with photosensitive biotin was significantly improved. Therefore, the sensitivity of the mushroom allergen IgE antibody detection kit was further improved by combining the use of photobiotin to couple mushroom allergens.
[0030] In addition, for the biotinylated mushroom allergen in the present invention, an MES system buffer containing 0.9% NaCl, 1% BSA, 1% glycerol, 0.01% Triton X-100, and 0.1% Proclin-300 is selected for use with the biotinylated mushroom allergen preparation reagent of the present invention, and it can be used to dilute the sample to be tested to ensure the detection sensitivity and the accuracy of the detection result.
[0031] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.
[0032] In the following examples and comparative examples, the unspecified implementation conditions are conventional conditions in the industry, and the unspecified reagents are all commercially available products. The instrument used for detection is the SMART 6500HOB fully automatic chemiluminescence immunoassay analyzer of Chongqing Cosmay Biotechnology Co., Ltd.
[0033] Example 1
[0034] The present embodiment provides a mushroom allergen IgE antibody detection kit, which includes biotinylated mushroom allergens, streptavidin-labeled nanomagnetic particles, alkaline phosphatase-labeled anti-human IgE secondary antibody, sample diluent, and luminescent substrate.
[0035] The biotinylated mushroom allergen was prepared as follows:
[0036] 1. Pre-treat purchased mushroom allergens, including the following steps:
[0037] 1.1 Dissolve the gel filtration resin according to the manufacturer's instructions and load it into the chromatographic column, ensuring that the resin is fully wetted and that no bubbles are present.
[0038] 1.2 Wash the column with MES buffer and equilibrate the resin.
[0039] 1.3 Slowly load the sample into the column.
[0040] 1.4 Elute the sample using MES buffer.
[0041] 1.5 Collect the eluate and fractionate it according to the elution curve.
[0042] 1.6 Use SDS-PAGE to detect the purity and amount of protein in the fraction, and select the fraction with a molecular weight less than 50 kDa for concentration.
[0043] 1.7 Heat the water bath to above 80°C, heat the purified mushroom allergens with a molecular weight of less than 50 kDa for 10 minutes, take them out, cool them to room temperature and store them in a refrigerator.
[0044] The electrophoresis results of mushroom allergens before and after pretreatment are shown in Figure 1 , WES analysis results are shown in Figure 2 . Figure 1 It shows that proteins greater than or equal to 50 kDa in the pre-treated mushroom allergens have been basically processed. Figure 2It showed that the mushroom allergen before pretreatment had an obvious reaction peak with Cladosporium at 66KDa, and the protein at the 66KDa position of the mushroom allergen after pretreatment had no reactivity.
[0045] 2. Perform biotin conjugation on the pretreated mushroom allergens, including the following steps:
[0046] 2.1 Add 20 μL of the pretreated mushroom allergens into the centrifuge tube.
[0047] 2.2 Add an equal volume of photosensitive biotin acetate (CAS No. 96087-38-6) in a dark environment and mix well.
[0048] 2.3 Place the mixture in a dark room and irradiate with a light marker lamp for 30 minutes.
[0049] 2.4 Add pure water to a total volume of 100 μL.
[0050] 2.5 Add 100 μL of sec-butanol, mix well, centrifuge at 10000 rpm for 10 min, remove the supernatant, add 100 μL of sec-butanol, centrifuge, and concentrate the aqueous phase to 30 μL.
[0051] 2.6 Add 4 μL 3M NaAc, mix well, then add 2 times the volume of anhydrous ethanol, mix well, and incubate at -20°C overnight.
[0052] 2.7 Centrifuge at 15000 rpm for 15 min at 4°C and collect the precipitate.
[0053] 2.8 Vacuum drying to obtain biotinylated mushroom allergen.
[0054] The streptavidin-labeled nanomagnetic particles and the alkaline phosphatase-labeled anti-human IgE secondary antibody were from the kit with the catalog number HY00004 of Hunan Haooubo Biopharmaceutical Co., Ltd.
[0055] Allergen diluent: MES buffer (pH 6.0±0.2) containing 0.9wt% NaCl, 1wt% BSA, 1% v / v glycerol, 0.01% v / v Triton X-100, and 0.1% v / v Proclin-300.
[0056] The luminescent substrate is a product of Jiangsu Haooubo Biopharmaceutical Co., Ltd., catalog number: MY00073.
[0057] The cleaning fluid is a product of Jiangsu Haooubo Biopharmaceutical Co., Ltd., item number: MY00071.
[0058] Comparative Example 1
[0059] This example provides another mushroom allergen IgE antibody detection kit, which is basically the same as Example 1, except that the preparation method of the biotinylated mushroom allergen is different. In this comparative example, the biotinylated mushroom allergen is directly obtained by biotin coupling with purchased mushroom allergens, and the other reagents are the same as Example 1.
[0060] Comparative Example 2
[0061] This example provides another mushroom allergen IgE antibody detection kit, which is basically the same as Example 1, except that the preparation method of the biotinylated mushroom allergen is different, and the other reagents are the same as Example 1. In this comparative example, when preparing the biotinylated mushroom allergen, only the part with a molecular weight greater than or equal to 50 kDa is removed during the pretreatment of the purchased mushroom allergen, and no heating treatment is performed.
[0062] Performance Testing
[0063] The mushroom allergen IgE antibody detection kits of the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2 and the existing commercially available mushroom allergen IgE antibody detection kit (manufacturer: Phadia, item number: 14-5086-01) were used to detect mushroom allergen IgE antibodies on 15 clinically negative samples of mushroom allergen IgE antibodies and 35 clinically positive samples of mushroom allergen IgE antibodies, respectively. At the same time, the commercially available Cladosporium allergen IgE antibody detection kit (manufacturer: Phadia, item number: 14-4105-01) was used to detect the Cladosporium antibodies of each clinical sample. The Cladosporium antibody detection method is referred to the instruction manual, and the use method of the commercially available mushroom allergen IgE antibody detection kit is referred to its instruction manual. The detection method of the mushroom allergen IgE antibody detection kits of the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2 is as follows:
[0064] Step 1: Add the biotinylated mushroom allergen to the allergen diluent in an appropriate amount to prepare a mushroom allergen reagent. After multiple tests, it was determined that the concentration of the mushroom allergen reagent of Example 1 was prepared to be 1.5 μg / mL, the concentration of the mushroom allergen reagent of Comparative Example 1 was prepared to be 10 μg / mL, and the concentration of the mushroom allergen reagent of Comparative Example 2 was prepared to be 2 μg / mL. Later, the biotinylated mushroom allergen can be diluted to a suitable concentration using the allergen diluent as an allergen reagent and stored at 4°C for later use.
[0065] Step 2: Pipette 15 μL of the sample to be tested, 15 μL of the allergen reagent, and 45 μL of streptavidin-labeled nanomagnetic particles, mix them, and react at 37°C for 15 minutes.
[0066] Step 3: Wash three times with washing solution, 600 μL each time.
[0067] Step 4: Add 130 μL of alkaline phosphatase-labeled anti-human IgE secondary antibody and react at 37°C for 15 minutes.
[0068] Step 5: Clean as in step 3.
[0069] Step 6: Finally, add 200 μL of luminescent substrate and react at 37°C for 5 minutes.
[0070] Step 7: Read the luminescence value and use the corresponding curve to calculate the concentration value.
[0071] The curve is obtained by detecting different concentrations of standard products using the above kit, and plotting the detection concentration and luminescence value.
[0072] The test results are summarized in Table 1.
[0073] Table 1
[0074]
[0075]
[0076] Table 1 shows the ImmunoCAP values in IU / mL. The test results of each kit are considered negative when the value is less than 0.35 IU / mL, and positive when the value is greater than or equal to 0.35 IU / mL.
[0077] Table 1 shows that 5 of the 15 clinically negative samples of mushroom allergen IgE antibodies were positive for Cladosporium antibodies. When the kit of Example 1 was used for detection, all 15 samples were detected as negative for mushroom allergen IgE antibodies. When the kit of Comparative Example 1 was used for detection, all 5 Cladosporium antibody-positive samples were detected as positive for mushroom allergen IgE antibodies. When the kit of Comparative Example 2 was used for detection, 2 Cladosporium antibody-positive samples were detected as positive for mushroom allergen IgE antibodies. When the commercially available mushroom allergen IgE antibody detection kit was used for detection, all 5 Cladosporium antibody-positive samples were detected as positive for mushroom allergen IgE antibodies.
[0078] Table 2 shows that when the kit of Example 1 was used to detect 35 clinically positive samples of mushroom allergen IgE antibodies, only positive sample 4 was detected as negative for mushroom allergen IgE antibodies, and the rest were positive for mushroom allergen IgE antibodies. When the kit of Comparative Example 1 was used to detect 35 clinically positive samples of mushroom allergen IgE antibodies, 15 samples were detected as negative for mushroom allergen IgE antibodies. When the kit of Comparative Example 2 was used to detect 35 clinically positive samples of mushroom allergen IgE antibodies, 1 sample was detected as negative for mushroom allergen IgE antibodies. When the commercially available mushroom allergen IgE antibody detection kit was used to detect 35 clinically positive samples of mushroom allergen IgE antibodies, 5 samples were detected as negative for mushroom allergen IgE antibodies.
[0079] The test results of the above kit were compared with the clinical results, and the negative and positive coincidence rates were statistically shown in Table 2.
[0080] Table 2
[0081]
[0082] The above results show that the kit of Example 1 has higher sensitivity and cleaner background, which can effectively avoid the phenomenon of misjudgment caused by cross-talk between mushroom and mold allergens. The detection results of clinical samples are more accurate and more consistent with clinical diagnosis. In addition, the biotinylation efficiency of mushroom allergens in Example 1 is higher.
[0083] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.
Claims
1. A mushroom allergen IgE antibody detection kit, comprising biotinylated mushroom allergens, streptavidin-labeled nanomagnetic particles, alkaline phosphatase-labeled anti-human IgE secondary antibody, sample diluent and luminescent substrate, characterized in that: The biotinylated mushroom allergen is a mushroom allergen that is labeled with photosensitive biotin and has a molecular weight of less than 50 kDa and has been subjected to a heat treatment.
2. The mushroom allergen IgE antibody detection kit according to claim 1, characterized in that: The preparation method of the mushroom allergen comprises: removing the protein component with a molecular weight greater than or equal to 50 kDa from the mushroom allergen by gel filtration chromatography, and heating the remaining components in a water bath at 80° C. or above to obtain the mushroom allergen.
3. The mushroom allergen IgE antibody detection kit according to claim 2, characterized in that: The temperature of the water bath heating does not exceed 100°C.
4. The mushroom allergen IgE antibody detection kit according to claim 2, characterized in that: The water bath heating time is 8 to 15 minutes.
5. The mushroom allergen IgE antibody detection kit according to claim 1, characterized in that: The mushroom allergen IgE antibody detection kit also includes an allergen diluent, which is a MES system buffer containing 0.8-1.0wt% NaCl, 0.8-1.5wt% BSA, 0.8-1.5v / v% glycerol, 0.005-0.02v / v% surfactant, and 0.05-0.2v / v% preservative.
6. The mushroom allergen IgE antibody detection kit according to claim 5, characterized in that: The surfactant is Triton X-100, and the preservative is Proclin-300.
7. The mushroom allergen IgE antibody detection kit according to claim 1, characterized in that: The photosensitive biotin is photosensitive biotin acetate.
8. The mushroom allergen IgE antibody detection kit according to claim 1, characterized in that: The mushroom allergen before treatment was the mushroom allergen with the product number XPF150D3A2.5 from Stallergenes Greer.
9. The mushroom allergen IgE antibody detection kit according to claim 1, characterized in that: The solid content of the streptavidin-labeled nanomagnetic particles is 0.1-1 mg / mL, and the particle size of the nanomagnetic particles is 0.5-1.5 μm; and / or, the concentration of the alkaline phosphatase-labeled anti-human IgE secondary antibody is 0.1-1 μg / ml; And / or, the luminescent substrate is AMPPD.
10. The mushroom allergen IgE antibody detection kit according to claim 1, characterized in that: The biotinylated mushroom allergen is stored in a solid state after vacuum drying, or the biotinylated mushroom allergen is mixed with the allergen diluent to prepare a mushroom allergen reagent with a concentration of 1 to 5 μg / mL for storage.