CN118624911A - A milk component allergen-specific IgE detection kit and method thereof - Google Patents
A milk component allergen-specific IgE detection kit and method thereof Download PDFInfo
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- CN118624911A CN118624911A CN202410745046.XA CN202410745046A CN118624911A CN 118624911 A CN118624911 A CN 118624911A CN 202410745046 A CN202410745046 A CN 202410745046A CN 118624911 A CN118624911 A CN 118624911A
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- biotin
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- lactoglobulin
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Abstract
本发明公开一种牛奶组分过敏原特异性IgE检测试剂盒及其方法,属于检测试剂盒技术领域。所述检测试剂盒包括:链霉亲和素包被的磁微粒、生物素标记的牛奶组分过敏原、碱性磷酸酶标记的鼠抗人IgE抗体、校准品、生物素标记的鼠抗人IgE抗体;其中,生物素标记的牛奶组分过敏原包括:分别用生物素标记的α‑乳清蛋白、β‑乳球蛋白、牛血清白蛋白、酪蛋白和重组β‑乳球蛋白。本发明将天然提取蛋白和重组蛋白结合,并对天然提取蛋白进行预先处理后进行标记,制备的牛奶过敏源检测试剂盒可以同时快速检测被测者对牛奶4种组分过敏情况,且能分析其对每一组分的具体致敏等级,可以解决患者检测牛奶过敏原不明晰,盲目拒食等的问题。
The present invention discloses a milk component allergen-specific IgE detection kit and method thereof, and belongs to the technical field of detection kits. The detection kit comprises: magnetic particles coated with streptavidin, milk component allergens labeled with biotin, mouse anti-human IgE antibodies labeled with alkaline phosphatase, calibrators, and mouse anti-human IgE antibodies labeled with biotin; wherein, the milk component allergens labeled with biotin include: α-lactalbumin, β-lactoglobulin, bovine serum albumin, casein, and recombinant β-lactoglobulin labeled with biotin, respectively. The present invention combines natural extract proteins with recombinant proteins, and pre-treats the natural extract proteins before labeling. The prepared milk allergen detection kit can simultaneously and rapidly detect the allergy of the test subject to the four components of milk, and can analyze the specific sensitization level of each component, which can solve the problems of patients not clearly detecting milk allergens and blindly refusing to eat.
Description
技术领域Technical Field
本发明涉及检测试剂盒技术领域,具体涉及一种牛奶组分过敏原特异性IgE检测试剂盒及其方法。The present invention relates to the technical field of detection kits, and in particular to a milk component allergen-specific IgE detection kit and a method thereof.
背景技术Background Art
食物过敏是当今重大的卫生学问题,对人们的身体健康、生活质量造成严重的影响,对食物过敏的致病机理、检测、防治的研究日益受到重视。牛奶过敏是食物过敏中的主要类型之一,在婴幼儿中高发,严重威胁患儿的健康和生长发育。牛奶是人类脂肪、蛋白质和微量营养素的最佳来源,也被认为是引起90%个体过敏的八组食物之一。过敏人群会出现皮肤瘙痒、红色皮疹、恶心、呕吐、腹泻、腹痛,以及急性呼吸等反应,严重者可导致死亡。各地发病率从1%到7.5%不等。在对中国的食物过敏儿童的研究中发现,牛奶过敏在自我报告型过敏原中排名第三,患病率约10.8%。大多数牛奶过敏的患者在3岁-5岁会产生耐受,不再受到牛奶过敏的困扰,然而仍有15%-20%的患者对牛奶过敏会持续更久。Food allergy is a major hygiene problem today, which has a serious impact on people's health and quality of life. The research on the pathogenic mechanism, detection, prevention and treatment of food allergy has received increasing attention. Milk allergy is one of the main types of food allergy, which is highly prevalent in infants and young children, and seriously threatens the health and growth and development of children. Milk is the best source of fat, protein and micronutrients for humans, and is also considered to be one of the eight groups of food that cause allergies in 90% of individuals. Allergic people will experience skin itching, red rash, nausea, vomiting, diarrhea, abdominal pain, and acute breathing, and severe cases can lead to death. The incidence rate ranges from 1% to 7.5% in various places. In a study of children with food allergies in China, it was found that milk allergy ranked third among self-reported allergens, with a prevalence of about 10.8%. Most patients with milk allergy will develop tolerance at the age of 3-5 years old and will no longer be troubled by milk allergies. However, 15%-20% of patients will still have longer-lasting milk allergies.
牛奶过敏原主要包括α-乳白蛋白(Bos d 4)、β-乳球蛋白(Bos d 5)、牛血清白蛋白(Bos d 6)、酪蛋白(Bos d 8)。目前,牛奶过敏原存在的主要问题是天然提取的原料抗原表位被掩盖,导致标记后的过敏原与IgE结合能力降低,过敏患者对牛乳蛋白的IgE反应活性具有很大的可变性,并且并不能确定导致牛乳致敏性是单一过敏原引起,还是多个过敏原共同引起。Milk allergens mainly include α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5), bovine serum albumin (Bos d 6), and casein (Bos d 8). At present, the main problem with milk allergens is that the antigenic epitopes of the naturally extracted raw materials are masked, resulting in a decrease in the ability of the labeled allergens to bind to IgE. The IgE reactivity of allergic patients to milk proteins has great variability, and it is not certain whether the milk allergy is caused by a single allergen or multiple allergens.
及早检测和确诊食物过敏,是该疾病防控的关键。食物过敏的致病机理主要为I型超敏反应,机体对某种食物过敏时,会在体内产生特异性抗体(主要为IgE型),能特异性结合食物中导致过敏的成分—过敏原。尽管关于食物过敏的诊断已有大量研究,但如何精准识别过敏原一直是困扰临床的重要问题。过敏原组分解析诊断可同时检测多种食物蛋白的组分特异性IgE(SIgE),使临床对食物过敏患者的管理更加精准。Early detection and diagnosis of food allergies are the key to the prevention and control of the disease. The pathogenic mechanism of food allergies is mainly type I hypersensitivity reaction. When the body is allergic to a certain food, specific antibodies (mainly IgE type) will be produced in the body, which can specifically bind to the ingredients in food that cause allergies - allergens. Although there have been a lot of research on the diagnosis of food allergies, how to accurately identify allergens has always been an important problem that plagues clinical practice. Allergen component analysis diagnosis can simultaneously detect component-specific IgE (SIgE) of multiple food proteins, making the clinical management of patients with food allergies more accurate.
目前,牛奶的体内过敏诊断和过敏原特异性免疫治疗主要是依靠天然的牛奶提取物。然而,牛奶提取物中包含了致敏成分和非致敏成分,可能引起交叉反应而导致诊断不准确;且无法明确对患者致敏的具体过敏原组分;更不能定量地反应过敏原组分的特异性IgE水平。At present, the diagnosis of milk allergy in vivo and allergen-specific immunotherapy mainly rely on natural milk extracts. However, milk extracts contain allergenic and non-allergenic components, which may cause cross-reactions and lead to inaccurate diagnosis; and it is impossible to identify the specific allergen components that sensitize the patient; let alone quantitatively reflect the specific IgE level of the allergen components.
免疫磁珠(简称磁珠)具有操作简便、分离效率高、较大的比表面积及良好的物理稳定性的优点,是一种性能优良的磁性分离载体,且是纯化、检测和定量分析复杂分析物的通用工具。在外加磁场的作用下,能够快速与底物液相分离,磁珠以其高效分离和富集作用在免疫分析反应中使抗原对磁珠上固定化抗体的影响显著降低。磁珠作为整个免疫反应和信号收集的载体具有显著优越性,磁珠比表面积大,能结合更多的蛋白分子;磁珠表面化学基团与蛋白形成共价偶联,相对于物理吸附更牢固,更稳定;磁珠均匀悬浮于反应溶液中,大大增加与样本中待测物接触面积,减少反应所需的样本量,同时更快达到反应动态平衡,加快反应速度,节省反应时间。磁珠结构一般由磁性内核、包裹在外的高分子涂层及功能基层构成。高分子涂层材料一般为聚苯乙烯、聚氯乙烯,主要用于稳定新形成的磁珠表面和防止磁珠聚集。但高分子涂层可以与多种活性物质结合,如抗原、抗体等。BSA、酪蛋白等动物来源的蛋白质常作为封闭剂,阻断磁珠本身与样本中的非特异性吸附。但是在牛奶过敏原检测中,BSA和酪蛋白是牛乳主要过敏原,在检测中采用BSA和酪蛋白对磁珠进行封闭,会导致漏检等问题出现。Immunomagnetic beads (magnetic beads for short) have the advantages of simple operation, high separation efficiency, large specific surface area and good physical stability. They are a magnetic separation carrier with excellent performance and a universal tool for purification, detection and quantitative analysis of complex analytes. Under the action of an external magnetic field, they can be quickly separated from the substrate liquid phase. With their efficient separation and enrichment, magnetic beads significantly reduce the influence of antigens on the immobilized antibodies on the magnetic beads in the immunoassay reaction. Magnetic beads have significant advantages as carriers for the entire immune reaction and signal collection. The magnetic beads have a large specific surface area and can bind more protein molecules; the chemical groups on the surface of the magnetic beads form covalent couplings with proteins, which are stronger and more stable than physical adsorption; the magnetic beads are evenly suspended in the reaction solution, greatly increasing the contact area with the analyte in the sample, reducing the amount of sample required for the reaction, and reaching the dynamic equilibrium of the reaction faster, accelerating the reaction speed, and saving reaction time. The structure of magnetic beads is generally composed of a magnetic core, a polymer coating wrapped outside, and a functional base layer. The polymer coating material is generally polystyrene and polyvinyl chloride, which are mainly used to stabilize the newly formed surface of the magnetic beads and prevent the aggregation of the magnetic beads. However, polymer coatings can bind to a variety of active substances, such as antigens, antibodies, etc. Animal-derived proteins such as BSA and casein are often used as blocking agents to block nonspecific adsorption of the magnetic beads themselves and the sample. However, in milk allergen detection, BSA and casein are the main allergens in cow's milk. Using BSA and casein to block the magnetic beads during the test will lead to problems such as missed detection.
对此,牛奶过敏原存在的主要问题是天然提取的原料抗原表位易被掩盖,导致生物素标记后的过敏原与IgE结合能力降低,过敏患者对牛乳蛋白的IgE反应活性具有很大的可变性,并且并不能确定导致牛乳致敏性是单一过敏原引起,还是多个过敏原共同引起。单纯使用过敏原组分组合会导致漏检,如专利CN 112748248A仅使用单独的α-乳清蛋白、β-乳球蛋白、牛血清白蛋白、记α-酪蛋白、β-酪蛋白和κ-酪蛋白,这些蛋白仅是天然的或者重组的均无法满足在临床的检测要求,天然提取蛋白会结合大量牛奶中其他物质,而导致与IgE结合表位被掩盖,仅使用重组蛋白而因其无天然蛋白的高级结构,这些均会导致漏检。In this regard, the main problem with milk allergens is that the antigen epitopes of naturally extracted raw materials are easily masked, resulting in a decrease in the ability of allergens to bind to IgE after biotin labeling. The IgE reactivity of allergic patients to milk protein has great variability, and it is not certain whether the milk allergy is caused by a single allergen or multiple allergens. The simple use of allergen component combinations will lead to missed detections. For example, patent CN 112748248A only uses separate α-lactalbumin, β-lactoglobulin, bovine serum albumin, α-casein, β-casein and κ-casein. These proteins, whether natural or recombinant, cannot meet the clinical detection requirements. Naturally extracted proteins will bind to a large number of other substances in milk, resulting in the masking of IgE binding epitopes. Only using recombinant proteins without the advanced structure of natural proteins will lead to missed detections.
在磁珠包被过程中,BSA、酪蛋白等动物来源的蛋白质常作为封闭剂,来阻断磁珠本身与样本中的非特异性吸附。但是在牛奶过敏原检测中,BSA和酪蛋白是牛乳主要过敏原,在检测中采用BSA和酪蛋白对磁珠进行封闭,会导致漏检或干扰等问题出现。In the process of magnetic bead coating, animal-derived proteins such as BSA and casein are often used as blocking agents to block the nonspecific adsorption of the magnetic beads themselves and the sample. However, in the detection of milk allergens, BSA and casein are the main allergens in cow milk. Using BSA and casein to block the magnetic beads in the test will lead to problems such as missed detection or interference.
此外,常用的免疫分析缓冲液buffer中均含有哺乳动物源性成分(牛、马、羊等血清或者BSA等),这些成分中牛的血清和牛血清白蛋白会造成严重的检测干扰,如CN112748248 A采用5%的马血清进行试剂配制,这会因交叉反应导致漏检。In addition, commonly used immunoassay buffers contain mammalian-derived components (cow, horse, sheep serum or BSA, etc.), of which cow serum and bovine serum albumin can cause serious detection interference. For example, CN112748248 A uses 5% horse serum to prepare reagents, which can lead to missed detection due to cross-reaction.
发明内容Summary of the invention
本发明的目的在于提供一种牛奶组分过敏原特异性IgE检测试剂盒及其方法,解决了天然提取的牛奶过敏原表位被掩盖,导致标记后的过敏原与IgE结合能力降低,过敏患者对牛乳蛋白的IgE反应活性具有很大的可变性,并且并不能确定导致牛乳致敏性是单一过敏原引起,还是多个过敏原共同引起的漏检;以及磁珠包被过程和采用缓冲剂因含有蛋白质造成检测干扰导致的漏检或干扰问题。The purpose of the present invention is to provide a milk component allergen-specific IgE detection kit and method thereof, which solves the problems that the naturally extracted milk allergen epitopes are masked, resulting in a reduced ability of the labeled allergen to bind to IgE, the IgE reactivity of allergic patients to milk protein has great variability, and it is impossible to determine whether the milk allergenicity is caused by a single allergen or a missed detection caused by multiple allergens; and the missed detection or interference problem caused by the magnetic bead coating process and the use of a buffer containing protein causing detection interference.
本发明通过下述技术方案实现:The present invention is achieved through the following technical solutions:
本发明一方面提供一种牛奶组分过敏原特异性IgE检测试剂盒,所述检测试剂盒包括:链霉亲和素包被的磁微粒、生物素标记的牛奶组分过敏原、碱性磷酸酶标记的鼠抗人IgE抗体、校准品、生物素标记的鼠抗人IgE抗体;The present invention provides a milk component allergen-specific IgE detection kit on the one hand, the detection kit comprising: magnetic particles coated with streptavidin, milk component allergens labeled with biotin, mouse anti-human IgE antibodies labeled with alkaline phosphatase, calibrators, and mouse anti-human IgE antibodies labeled with biotin;
其中,生物素标记的牛奶组分过敏原包括:生物素标记α-乳清蛋白、生物素标记β-乳球蛋白、生物素标记牛血清白蛋白、生物素标记酪蛋白和生物素标记重组β-乳球蛋白。Among them, biotin-labeled milk component allergens include: biotin-labeled α-lactalbumin, biotin-labeled β-lactoglobulin, biotin-labeled bovine serum albumin, biotin-labeled casein and biotin-labeled recombinant β-lactoglobulin.
进一步地,在所述的牛奶组分过敏原特异性IgE检测试剂盒中,所述α-乳清蛋白为α-亚麻酸诱导处理α-乳清蛋白;Furthermore, in the milk component allergen-specific IgE detection kit, the α-lactalbumin is α-linolenic acid-induced α-lactalbumin;
和/或,所述α-亚麻酸诱导处理α-乳清蛋白的制备包括:将α-乳白蛋白和α-亚麻酸按照摩尔比1:50混匀后,在温度30~60℃反应10~30min,经离心透析获得所述α-亚麻酸诱导处理α-乳清蛋白。And/or, the preparation of the α-linolenic acid induced α-lactalbumin comprises: mixing α-lactalbumin and α-linolenic acid at a molar ratio of 1:50, reacting at a temperature of 30 to 60° C. for 10 to 30 minutes, and obtaining the α-linolenic acid induced α-lactalbumin by centrifugal dialysis.
进一步地,在所述的牛奶组分过敏原特异性IgE检测试剂盒中,所述β-乳球蛋白为超声波处理β-乳球蛋白;Furthermore, in the milk component allergen-specific IgE detection kit, the β-lactoglobulin is ultrasonically treated β-lactoglobulin;
和/或,所述超声波处理β-乳球蛋白的制备包括:And/or, the preparation of ultrasonically treated β-lactoglobulin comprises:
在低于15℃温度下,将β-乳球蛋白以100~150W/cm2功率,超声处理10~40min。At a temperature below 15°C, the β-lactoglobulin is ultrasonically treated at a power of 100 to 150 W/ cm2 for 10 to 40 minutes.
进一步地,在所述的牛奶组分过敏原特异性IgE检测试剂盒中,所述重组β-乳球蛋白的氨基酸序列如SEQ ID NO:1所示。Furthermore, in the milk component allergen-specific IgE detection kit, the amino acid sequence of the recombinant β-lactoglobulin is as shown in SEQ ID NO:1.
进一步地,在所述的牛奶组分过敏原特异性IgE检测试剂盒中,所述链霉亲和素包被的磁微粒的浓度为1~10μg/mL;Further, in the milk component allergen-specific IgE detection kit, the concentration of the streptavidin-coated magnetic particles is 1 to 10 μg/mL;
和/或,所述生物素标记的牛奶组分过敏原的浓度为0.1~1μg/mL;and/or, the concentration of the biotin-labeled milk component allergen is 0.1 to 1 μg/mL;
和/或,所述碱性磷酸酶标记的鼠抗人IgE抗体的浓度为0.1~1μg/mL;and/or, the concentration of the alkaline phosphatase-labeled mouse anti-human IgE antibody is 0.1 to 1 μg/mL;
和/或,所述生物素标记的鼠抗人IgE抗体的浓度为0.1~1μg/mL。And/or, the concentration of the biotin-labeled mouse anti-human IgE antibody is 0.1-1 μg/mL.
进一步地,在所述的牛奶组分过敏原特异性IgE检测试剂盒中,所述链霉亲和素包被的磁微粒采用0.1~5% DB1130、0.1~1%吐温-20和100-200mM NaCl、pH=7-8的Tris-HCl缓冲液配制成悬浮液。Furthermore, in the milk component allergen-specific IgE detection kit, the streptavidin-coated magnetic particles are prepared into a suspension using 0.1-5% DB1130, 0.1-1% Tween-20 and 100-200 mM NaCl, pH=7-8 Tris-HCl buffer.
进一步地,在所述的牛奶组分过敏原特异性IgE检测试剂盒中,所述碱性磷酸酶标记的鼠抗人IgE抗体包括采用含1%鱼血清、pH=6-7的MES缓冲液配置成溶液。Furthermore, in the milk component allergen-specific IgE detection kit, the alkaline phosphatase-labeled mouse anti-human IgE antibody is configured into a solution using a MES buffer solution containing 1% fish serum and a pH of 6-7.
进一步地,在所述的牛奶组分过敏原特异性IgE检测试剂盒中,所述生物素标记的鼠抗人IgE抗体的制备包括:Furthermore, in the milk component allergen-specific IgE detection kit, the preparation of the biotin-labeled mouse anti-human IgE antibody comprises:
将生物素与鼠抗人IgE抗体混合,避光反应10~60min,加入0.1~5%Tris封闭,采用浓度为pH=7-8,0.1~1M磷酸盐缓冲液配制成溶液;The biotin and mouse anti-human IgE antibody are mixed, reacted in the dark for 10 to 60 minutes, 0.1 to 5% Tris is added for blocking, and a solution is prepared using a phosphate buffer solution with a concentration of pH = 7-8 and 0.1 to 1M;
和/或,所述生物素为BNHS-Biotin。And/or, the biotin is BNHS-Biotin.
进一步地,在所述的牛奶组分过敏原特异性IgE检测试剂盒中,所述生物素标记的牛奶组分过敏原的制备包括:Furthermore, in the milk component allergen-specific IgE detection kit, the preparation of the biotin-labeled milk component allergen includes:
将生物素与牛奶组分过敏原混合,避光反应10~60min,加入0.1~5%Tris封闭,采用浓度为pH=7-8,0.1~1M磷酸盐缓冲液配制成溶液;The biotin and milk component allergens are mixed, reacted in the dark for 10 to 60 minutes, 0.1 to 5% Tris is added for blocking, and a solution is prepared using a phosphate buffer solution with a concentration of pH = 7-8 and 0.1 to 1 M;
和/或,所述生物素为BNHS-Biotin。And/or, the biotin is BNHS-Biotin.
本发明第二方面提供一种牛奶组分过敏原特异性IgE检测方法,基于所述的牛奶组分过敏原特异性IgE检测试剂盒,所述检测方法包括:The second aspect of the present invention provides a method for detecting specific IgE of milk component allergens. Based on the milk component allergen specific IgE detection kit, the detection method comprises:
将待测样本与链霉亲和素包被的磁微粒悬浮液和生物素标记的牛奶组分过敏原混匀并在30~40℃孵育1-30min;The sample to be tested was mixed with a suspension of magnetic microparticles coated with streptavidin and a biotin-labeled milk component allergen and incubated at 30-40°C for 1-30 min;
经洗涤后,加入碱性磷酸酶标记的鼠抗人IgE抗体反应1~30min,形成固相抗原-抗体-酶标二抗复合物;经洗涤后,加入发光底物,测定化学发光强度。After washing, alkaline phosphatase-labeled mouse anti-human IgE antibody is added to react for 1 to 30 minutes to form a solid-phase antigen-antibody-enzyme-labeled secondary antibody complex; after washing, a luminescent substrate is added to measure the chemiluminescence intensity.
本发明与现有技术相比,具有如下的优点和有益效果:Compared with the prior art, the present invention has the following advantages and beneficial effects:
1、本发明提供的牛奶组分过敏原特异性IgE检测试剂盒及其方法,将天然提取蛋白和重组蛋白结合,并对天然提取蛋白进行预先处理后进行标记,制备的牛奶过敏源检测试剂盒可以同时快速检测被测者对牛奶4种组分过敏情况,且能分析其对每一组分的具体致敏等级,可以解决患者检测牛奶过敏原不明晰,盲目拒食等的问题。1. The milk component allergen-specific IgE detection kit and method provided by the present invention combine natural extracted proteins and recombinant proteins, and pre-treat the natural extracted proteins before labeling. The prepared milk allergen detection kit can simultaneously and quickly detect the subject's allergies to the four components of milk, and can analyze the specific sensitization level to each component, which can solve the problem of patients' unclear detection of milk allergens and blind refusal to eat.
2、本发明提供的牛奶组分过敏原特异性IgE检测试剂盒及其方法,通过对天然提取α-乳清蛋白采用α-亚麻酸诱导以改变蛋白结构,暴露出更多与IgE的结合位点,提高其与特异性IgE的结合能力;对天然提取β-乳球蛋白用超声波处理提高其抗原性;将天然蛋白和重组蛋白相结合,模拟在体内消化后的抗原表位,提升致敏性,提高检测准确性。2. The milk component allergen-specific IgE detection kit and method provided by the present invention use α-linolenic acid to induce naturally extracted α-lactalbumin to change the protein structure, expose more binding sites with IgE, and improve its binding ability with specific IgE; use ultrasonic treatment to improve the antigenicity of naturally extracted β-lactoglobulin; combine natural protein and recombinant protein to simulate the antigenic epitope after digestion in the body, enhance sensitization, and improve detection accuracy.
3、本发明提供的牛奶组分过敏原特异性IgE检测试剂盒及其方法,使用非动物源性封闭剂,可以有效避免动物源性成分造成的干扰,使检测结果更准确可靠。3. The milk component allergen-specific IgE detection kit and method provided by the present invention use a non-animal-derived blocking agent, which can effectively avoid interference caused by animal-derived ingredients and make the detection results more accurate and reliable.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本发明示例性实施方式的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。在附图中:In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:
图1为分离纯化后的α-乳清蛋白、β-乳球蛋白、酪蛋白和牛血清白蛋白的电泳图;FIG1 is an electrophoretic diagram of separated and purified α-lactalbumin, β-lactoglobulin, casein and bovine serum albumin;
图2为超声波处理时间对β-乳球蛋白分子量的影响结果;FIG2 is the effect of ultrasonic treatment time on the molecular weight of β-lactoglobulin;
图3为超声波处理时间对β-乳球蛋白与IgE结合能力的影响;FIG3 shows the effect of ultrasonic treatment time on the binding ability of β-lactoglobulin to IgE;
图4为重组β-乳球蛋白的电泳图;FIG4 is an electrophoretic diagram of recombinant β-lactoglobulin;
图5为β-乳球蛋白与IgE结合能力的比较;FIG5 is a comparison of the binding ability of β-lactoglobulin and IgE;
图6为α-亚麻酸诱导α-乳白蛋白的电泳图;FIG6 is an electrophoretic diagram of α-lactalbumin induced by α-linolenic acid;
图7为α-亚麻酸诱导α-乳白蛋白与IgE结合能力与Phadia测值的比较;FIG7 is a comparison of the ability of α-lactalbumin to bind to IgE induced by α-linolenic acid and the value measured by Phadia;
图8为BSA和商业阻断剂DB1130封闭裸磁珠的非特异性吸附样本的比较;FIG8 is a comparison of nonspecific adsorption samples of bare magnetic beads blocked by BSA and commercial blocking agent DB1130;
图9为不同封闭剂测试临床样本的比较。FIG. 9 is a comparison of clinical samples tested with different sealants.
具体实施方式DETAILED DESCRIPTION
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with Examples and accompanying drawings. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
实施例1:天然蛋白的提取和分离纯化Example 1: Extraction, separation and purification of natural protein
(一)总蛋白提取:(I) Total protein extraction:
新鲜牛奶5000rpm离心30min后,弃去上层脂肪,按1:5的体积比加入-20℃预冷丙酮,于4℃环境中浸泡去脂24h,其间搅拌2次,多次换液直至丙酮澄清。而后3000rpm离心30min,弃去丙酮,将沉淀置于超净通风橱,于4℃环境中通风挥干丙酮后称重,按8mL/g的比例溶于PBS中(0.01M,pH7.4),于4℃环境中提取24h,其间搅拌数次。最后以12000rpm低温冷冻离心20min,收集上清即为牛奶总蛋白粗提液。分装后于-20℃保存。After fresh milk was centrifuged at 5000rpm for 30min, the upper fat was discarded, and -20℃ pre-cooled acetone was added at a volume ratio of 1:5, and the milk was soaked in a 4℃ environment for 24h to remove fat, stirred twice, and the solution was changed several times until the acetone was clear. Then centrifuged at 3000rpm for 30min, the acetone was discarded, the precipitate was placed in an ultra-clean fume hood, ventilated and evaporated in a 4℃ environment, and the acetone was weighed, dissolved in PBS (0.01M, pH7.4) at a ratio of 8mL/g, and extracted in a 4℃ environment for 24h, stirring several times. Finally, centrifuged at 12000rpm for 20min, and the supernatant was collected as the crude extract of total milk protein. After aliquoting, store at -20℃.
提取出含α-乳白蛋白、β-乳球蛋白、牛血清白蛋白、酪蛋白的总蛋白。The total protein including α-lactalbumin, β-lactoglobulin, bovine serum albumin and casein was extracted.
(二)β-乳球蛋白和α-乳白蛋白分离纯化(II) Separation and purification of β-lactoglobulin and α-lactalbumin
(1)硫酸铵盐析乳清蛋白(1) Ammonium sulfate salting out whey protein
采用50%和90%饱和度的硫酸分级盐析沉淀牛乳清蛋白。乳清中加入等体积的饱和硫酸铵溶液(硫酸铵饱和度变为0%),磁力搅拌30min后4℃静置过夜,8000rpm离心30min,分离沉淀和上清,沉淀用PBS(0.01M,pH6.8)复溶,而上清液继续用90%饱和度的硫酸铵盐析,取100ml上清液,计算并称量出该体积下由50%饱和度提高到90%饱和度需要补充的硫酸铵的量(查硫酸铵饱和度表),在磁力搅拌的情况下将硫酸铵缓慢加入上清液中,继续磁力搅拌30min后4℃静置过夜,8000rpm离心30min取沉淀用PBS复溶。The bovine whey protein was precipitated by graded salting out of sulfuric acid with 50% and 90% saturation. An equal volume of saturated ammonium sulfate solution (ammonium sulfate saturation becomes 0%) was added to the whey, and magnetic stirring was continued for 30 minutes and then left to stand overnight at 4°C, centrifuged at 8000rpm for 30 minutes, and the precipitate and the supernatant were separated. The precipitate was redissolved with PBS (0.01M, pH 6.8), and the supernatant was further salted out with ammonium sulfate with 90% saturation. 100 ml of the supernatant was taken, and the amount of ammonium sulfate required to be supplemented from 50% saturation to 90% saturation under this volume was calculated and weighed (check the ammonium sulfate saturation table), and ammonium sulfate was slowly added to the supernatant under magnetic stirring, and magnetic stirring was continued for 30 minutes and then left to stand overnight at 4°C, centrifuged at 8000rpm for 30 minutes, and the precipitate was redissolved with PBS.
(2)凝胶层析分离乳清蛋白(2) Gel chromatography to separate whey protein
选用Sephadex G 25、Sephadex G 100和Sephadex G 200凝胶对浓缩乳清和硫酸铵盐析组分进行层析纯化。Sephadex G 25, Sephadex G 100 and Sephadex G 200 gels were used for chromatography purification of concentrated whey and ammonium sulfate salting-out fractions.
①凝胶的预处理:采用水浴法,称取一定量的凝胶粉末溶于足量超纯水中沸水浴使凝胶充分溶胀,再用超纯水浮洗若干次去除细碎颗粒,泡于凝胶储存液中于4℃保存备用。① Pretreatment of gel: Using the water bath method, weigh a certain amount of gel powder and dissolve it in sufficient ultrapure water in a boiling water bath to fully swell the gel, then float and wash it with ultrapure water several times to remove fine particles, and soak it in gel storage solution and store it at 4°C for later use.
②装柱:将溶胀后的凝胶在磁力搅拌下用真空泵脱气处理,而后进行装柱选择柱高约80cm,缓冲液为0.01MPBS,凝胶沉降完全后剪一圆形滤纸盖于胶面以缓冲液流。而后连接监测装置和横流泵,泵速20rpm,用PBS充分平衡② Column loading: Degas the swollen gel with a vacuum pump under magnetic stirring, and then load the column. Select a column height of about 80cm, and use 0.01M PBS as the buffer. After the gel has settled completely, cut a round filter paper and cover the gel surface to allow the buffer to flow. Then connect the monitoring device and the cross-flow pump, pump speed 20rpm, and fully balance with PBS.
③上样及洗脱:将盐析产物和乳清样品分别进行上样层析。乳清采用millipore超滤器,以截流分子量10kDa的YM-10滤膜对乳清进行正压超滤浓缩将蛋白浓度调整为5-10mg/ml,一次上样2-3ml,用PBS洗脱,观测层析图谱分段收集各峰的洗脱组分。③ Loading and elution: The salting out product and whey sample were loaded for chromatography respectively. The whey was concentrated by positive pressure ultrafiltration using a millipore ultrafilter and a YM-10 filter membrane with a molecular weight cutoff of 10kDa to adjust the protein concentration to 5-10mg/ml. 2-3ml was loaded at a time, eluted with PBS, and the eluted components of each peak were collected in sections after observing the chromatogram.
④凝胶回收:收集完毕后继续用PBS将图谱冲至基线后回收凝胶,凝胶可在短期内重复使用数次纯化相同蛋白。④ Gel recovery: After collection, continue to use PBS to flush the spectrum to the baseline and then recover the gel. The gel can be reused several times in a short period of time to purify the same protein.
⑤凝胶的再生:依次用0.05M的NaOH抽滤数次,超纯水洗至中性,再用0.05M的硫酸抽滤数次,超纯水洗至中性,存于凝胶储存液中。⑤ Regeneration of gel: Filter several times with 0.05M NaOH, wash with ultrapure water until neutral, then filter several times with 0.05M sulfuric acid, wash with ultrapure water until neutral, and store in gel storage solution.
⑥将各洗脱组分测定蛋白含量,浓度太低者通过超滤浓缩,取样进行SDS-PAGE,观察分离效果。⑥ Determine the protein content of each eluted component. If the concentration is too low, concentrate it by ultrafiltration, take samples for SDS-PAGE, and observe the separation effect.
(3)离子交换层析分离β-乳球蛋白和α-乳白蛋白。(3) Separation of β-lactoglobulin and α-lactalbumin by ion exchange chromatography.
将浓缩乳清经凝胶层析后得到的只含有14kDa和18kDa蛋白的组分再次超滤浓缩后进行离子交换层析,选择DEAESephadexA50凝胶,水浴法溶胀处理后用真空泵脱气,装柱的同时用PBS(0.01M pH 6.8)充分平衡,控制柱高约20cm,柱顶放一滤纸片缓冲液流,上样量约2-3mL,先用PBS洗脱穿流峰,冲至基线后用含浓度为0.1M-1MNaCl的PBS进行梯度洗脱,收集各峰组分,分别测定蛋白含量,浓度太低者通过超滤浓缩。The fraction containing only 14kDa and 18kDa proteins obtained after gel chromatography of the concentrated whey was ultrafiltered and concentrated again, and then subjected to ion exchange chromatography. DEAE Sephadex A50 gel was selected, and it was swollen by water bath method and then degassed with a vacuum pump. While filling the column, PBS (0.01M pH 6.8) was used for full balance. The column height was controlled to be about 20cm, and a filter paper buffer was placed on the top of the column. The sample volume was about 2-3mL. The flow-through peak was first eluted with PBS, and after reaching the baseline, PBS containing 0.1M-1M NaCl was used for gradient elution. The peak components were collected and the protein content was determined respectively. Those with too low concentration were concentrated by ultrafiltration.
(三)酪蛋白的分离和纯化(III) Separation and purification of casein
采用等电点沉淀的方法分离牛乳酪蛋白,牛乳500mL在2500×g,20℃条件下离心30min,去除上层脂肪后得脱脂乳。10%醋酸调脱脂乳的pH为4.6,室温静置30min后,混合液在1000×g条件下离心20min,得沉淀为酪蛋白。酪蛋白用蒸馏水洗3次后再分散到蒸馏水中,并用0.5mol/L NaOH调pH 7.0,酪蛋白溶解后再用10%醋酸调pH为4.6,重新沉淀酪蛋白。此步骤重复3次后,酪蛋白于-20℃保存备用。The isoelectric precipitation method was used to separate bovine milk casein. 500 mL of bovine milk was centrifuged at 2500×g and 20°C for 30 minutes, and the upper fat was removed to obtain skim milk. The pH of the skim milk was adjusted to 4.6 with 10% acetic acid. After standing at room temperature for 30 minutes, the mixture was centrifuged at 1000×g for 20 minutes to obtain casein. The casein was washed 3 times with distilled water and then dispersed in distilled water, and the pH was adjusted to 7.0 with 0.5 mol/L NaOH. After the casein was dissolved, the pH was adjusted to 4.6 with 10% acetic acid to reprecipitate the casein. This step was repeated 3 times, and the casein was stored at -20°C for later use.
蛋白质快速纯化系统(FPLC)包括AKTAexporerAir100、填充20mL Cellulose DE-52(Pharmacia)介质的层析柱(Φ1.6cm×15cm),紫外检测波长λ=280nm。缓冲液A为pH7.0的咪唑-盐酸缓冲液,含0.01mol/L咪唑、3.3mol/L尿素、0.2%(体积分数)2-巯基乙醇。缓冲液B为在缓冲液A中加0.4%(质量分数)的NaCl。洗脱液流速为80mL/h,线性洗脱。粗分离得到的酪蛋白溶于起始缓冲液中,0.45μm滤膜过滤后上样,上样量为0.5mL,FPLC得到的酪蛋白和乳清蛋白经超纯水透析去盐、去尿素并经PEG-2000浓缩后制得酪蛋白。The protein rapid purification system (FPLC) includes AKTAexporerAir100, a chromatography column (Φ1.6cm×15cm) filled with 20mL Cellulose DE-52 (Pharmacia) medium, and an ultraviolet detection wavelength λ=280nm. Buffer A is an imidazole-hydrochloric acid buffer with a pH of 7.0, containing 0.01mol/L imidazole, 3.3mol/L urea, and 0.2% (volume fraction) 2-mercaptoethanol. Buffer B is 0.4% (mass fraction) NaCl added to buffer A. The eluent flow rate is 80mL/h, and the elution is linear. The casein obtained by crude separation is dissolved in the starting buffer, filtered through a 0.45μm filter membrane, and loaded with a sample volume of 0.5mL. The casein and whey protein obtained by FPLC are dialyzed against ultrapure water to remove salt, remove urea, and concentrate with PEG-2000 to obtain casein.
(四)牛血清白蛋白(BSA)分离纯化(IV) Separation and purification of bovine serum albumin (BSA)
将牛乳在在低温离心机(4℃),5000r/min,离心30min后将乳脂层去除,即得脱脂乳。脱脂乳在室温下用1M的HCl调至pH 4.3,40℃水浴20min,8000r/min离心20min(4℃)去除酪蛋白后,收集上清液乳清。将收集的乳清通过0.22μm过滤器过滤,-20℃保存备用。The milk was centrifuged in a low-temperature centrifuge (4°C) at 5000r/min for 30 minutes, and the fat layer was removed to obtain skim milk. The skim milk was adjusted to pH 4.3 with 1M HCl at room temperature, and then the skim milk was water bathed at 40°C for 20 minutes. After centrifugation at 8000r/min for 20 minutes (4°C) to remove casein, the supernatant whey was collected. The collected whey was filtered through a 0.22μm filter and stored at -20°C for later use.
采用DEAE-FF层析柱(16×25mm)对牛乳乳清进行离子交换层析。先用20mmol/L pH8.4Tris-HCl缓冲液平衡层析柱直至紫外吸收基线、电导稳定。牛乳乳清经透析膜透析过夜后上柱;用20mmol/L Tris-HCl缓冲液(pH8.4)充分冲洗非结合蛋白;待UV值较稳定,用一定梯度0.1~0.4M NaCl的20mmol/L Tris-HCl(pH8.4)缓冲液进行线性梯度洗脱。洗脱速度60mL/h,280nm下自动检测并记录。每管2mL收集馏分,将收集的洗脱峰-20℃保存备用。Ion exchange chromatography of bovine milk whey was performed using a DEAE-FF column (16×25mm). The column was first equilibrated with 20mmol/L pH8.4 Tris-HCl buffer until the UV absorption baseline and conductivity were stable. The bovine milk whey was dialyzed overnight with a dialysis membrane and then loaded onto the column; non-bound proteins were fully washed with 20mmol/L Tris-HCl buffer (pH8.4); when the UV value was relatively stable, a linear gradient elution was performed with a certain gradient of 0.1-0.4M NaCl in 20mmol/L Tris-HCl (pH8.4) buffer. The elution rate was 60mL/h, and the sample was automatically detected and recorded at 280nm. 2mL fractions were collected in each tube, and the collected elution peaks were stored at -20℃ for later use.
用含有20mmol/L Tris-HCl(pH 8.4)缓冲液平衡Sephacryl S-100柱至基线稳定。以相同的缓冲液进行洗脱,速度30mL/h,280nm下检测并记录。每管2mL收集馏分,将收集的洗脱峰-20℃保存。Equilibrate the Sephacryl S-100 column with 20mmol/L Tris-HCl (pH 8.4) buffer until the baseline is stable. Elute with the same buffer at a rate of 30mL/h, detect and record at 280nm. Collect 2mL fractions in each tube and store the collected elution peak at -20℃.
对分离纯化后的α-乳清蛋白、β-乳球蛋白、酪蛋白和牛血清白蛋白经过SDS-PAGE检测,各个洗脱峰结果如图1所示.The separated and purified α-lactalbumin, β-lactoglobulin, casein and bovine serum albumin were detected by SDS-PAGE, and the results of each elution peak are shown in Figure 1.
从图1可知,α-乳清蛋白分子量主要在14kDa附近,从图中可观察到条带1的主要聚集在14kDa左右,牛乳α-乳白蛋白纯度高达95%。根据前人研究结果发现β-乳球蛋白的分子量在18kDa附近,从图中可观察到条带2的主要聚集在18kDa,其纯度高达93%,能够为后续实验提供很好的保证。As shown in Figure 1, the molecular weight of α-lactalbumin is mainly around 14kDa, and it can be observed from the figure that band 1 is mainly concentrated at around 14kDa, and the purity of bovine milk α-lactalbumin is as high as 95%. According to previous research results, the molecular weight of β-lactoglobulin is around 18kDa, and it can be observed from the figure that band 2 is mainly concentrated at 18kDa, and its purity is as high as 93%, which can provide a good guarantee for subsequent experiments.
脱脂乳经分离纯化后,得到酪蛋白和牛血清白蛋白,文献报道酪蛋白和牛血清白蛋白分子量分别为30、66kD,SDS-PAGE测试结果如图所示,酪蛋白的分布在27-32kDa之间,牛血清白蛋白主要聚集在66kDa,且酪蛋白和牛血清白蛋白的纯度均>90%。After separation and purification of skim milk, casein and bovine serum albumin are obtained. Literature reports that the molecular weights of casein and bovine serum albumin are 30 and 66 kD, respectively. The SDS-PAGE test results are shown in the figure. The distribution of casein is between 27-32 kDa, and bovine serum albumin is mainly concentrated at 66 kDa. The purity of casein and bovine serum albumin is >90%.
实施例2:蛋白的处理和重组Example 2: Protein processing and reconstitution
(一)超声波处理β-乳球蛋白(I) Ultrasonic treatment of β-lactoglobulin
将天然β-乳球蛋白溶于双蒸馏水中,浓度为2mg/mL,将10mLβ-乳球蛋白溶液倒入25mL烧杯中,通过配备有3mm微尖探头的探头超声仪以120W/cm2的实际超声强度处理溶液20min。整个处理过程在冰浴条件下进行,以确保样品温度(低于15℃)。Natural β-lactoglobulin was dissolved in double distilled water at a concentration of 2 mg/mL, 10 mL of the β-lactoglobulin solution was poured into a 25 mL beaker, and the solution was treated for 20 min at an actual ultrasonic intensity of 120 W/cm2 using a probe sonicator equipped with a 3 mm micro-tip probe. The entire treatment process was carried out in an ice bath to ensure the sample temperature (below 15°C).
(二)α-亚麻酸诱导α-乳白蛋白以改变蛋白结构,提升其抗原性(ii) α-linolenic acid induces α-lactalbumin to change the protein structure and enhance its antigenicity
α-乳白蛋白、α-亚麻酸按照摩尔比1:50的条件下进行涡旋混匀,放入45℃的电热恒温水槽中反应20min。反应结束后,9000g离心20min,小心除去未结合的脂肪酸。为了进一步去除未结合的脂肪酸,将混合液移入透析袋中于4℃持续搅拌透析2天,每天更换3次超纯水,透析结束后,将获得的脂肪酸-α-乳白蛋白复合物分装,并于-20℃保存待用。α-lactalbumin and α-linolenic acid were vortexed and mixed at a molar ratio of 1:50, and placed in a 45°C electric thermostatic water bath for reaction for 20 minutes. After the reaction, centrifuge at 9000g for 20 minutes to carefully remove unbound fatty acids. In order to further remove unbound fatty acids, the mixed solution was transferred into a dialysis bag and continuously stirred and dialyzed at 4°C for 2 days, with ultrapure water replaced 3 times a day. After the dialysis, the obtained fatty acid-α-lactalbumin complex was packaged and stored at -20°C for use.
(三)重组β-乳球蛋白多肽(III) Recombinant β-lactoglobulin polypeptide
常用的表位预测软件包括:Commonly used epitope prediction software includes:
DNAStar protean system、Bepipred Linear Epitope Prediction system(BLEP)、Bioinformatics Predicted Antigenic Peptides(BPAP)systemDNAStar protean system, Bepipred Linear Epitope Prediction system (BLEP), Bioinformatics Predicted Antigenic Peptides (BPAP) system
其中DNAStar protean system以氨基酸序列的亲水性和抗原性作为表位预测的参数。选择亲水性好、柔韧性高、表面可及性好、抗原指数高的肽段作为进一步研究的候选表位,该肽段的氨基酸序列表如SEQ ID NO:1所示,见表1。综合以上三种生物信息学工具的β-乳球蛋白表位预测结果和参考已有的文献,确定出β-乳球蛋白候选表位,然后人工合成β-乳球蛋白多肽片段。The DNAStar protean system uses the hydrophilicity and antigenicity of the amino acid sequence as parameters for epitope prediction. A peptide with good hydrophilicity, high flexibility, good surface accessibility and high antigenic index was selected as a candidate epitope for further study. The amino acid sequence of the peptide is shown in SEQ ID NO: 1, see Table 1. Based on the β-lactoglobulin epitope prediction results of the above three bioinformatics tools and reference to existing literature, the candidate epitope of β-lactoglobulin was determined, and then the β-lactoglobulin polypeptide fragment was artificially synthesized.
表1重组β-乳球蛋白的氨基酸序列Table 1 Amino acid sequence of recombinant β-lactoglobulin
实施例3:试剂盒的制备Example 3: Preparation of kit
(1)链霉亲和素包被的磁微粒混悬液的配制(1) Preparation of Streptavidin-coated Magnetic Microparticle Suspension
称取20mg Tosyl磁珠到一支干净的管中;加入1000μL超纯水,涡旋10-30s,并置于水浴超声5min 2次或杆状超声10-30s 2次;置于磁力架吸附1分钟,移走上清液,再重复清洗一次,计算链霉亲和素的用量(最终5μg/mg)。Weigh 20 mg of Tosyl magnetic beads into a clean tube; add 1000 μL of ultrapure water, vortex for 10-30 seconds, and place in a water bath for 5 minutes twice or rod sonication for 10-30 seconds twice; place on a magnetic stand for adsorption for 1 minute, remove the supernatant, repeat the wash once, and calculate the amount of streptavidin (final 5 μg/mg).
取0.1M PBS缓冲溶液(PH 7.4)加入一支干净的管中,加入0.1mg链霉亲和素,使二者体积为600μL;涡旋混匀5s;加入400μL 0.1M PBS缓冲液(pH7.4溶解3M硫酸铵溶液);涡旋混匀5s,并放在滚动混匀仪上37℃滚动孵育2-4h,孵育完成后,置于磁力架1min直至磁珠溶液变澄清,移走上清液;1000μL100mM甘氨酸溶液(pH11.3)洗磁珠1遍:加入1000μL100mM甘氨酸溶液(pH11.3),涡旋5s,置于磁力架1min直至磁珠溶液变澄清,移走上清液;1000μL200mM甘氨酸溶液(pH2.8)洗磁珠1遍:加入1000μL 200mM甘氨酸溶液(pH2.8),涡旋5s,置于磁力架1min直至磁珠溶液变澄清,移走上清液;1000μL50mM Tris-HCl(pH7.4含140mMNaCl,1% DB1130和0.1%吐温-20);洗磁珠2遍:第1遍:加入1000μL 50mM Tris-HCl(pH7.4含140mM NaCl,1%DB1130和0.1%吐温-20),置于滚轴混匀仪上混合10min,再置于磁力架1min直至磁珠溶液变澄清,移走上清液;第2遍:加入1000μL50mM Tris-HCl(pH7.4含140mMNaCl,1%商业阻断剂DB1130和0.1%吐温-20),置于滚轴混匀仪上37℃混合过夜,再置于磁力架1min直至磁珠溶液变澄清,移走上清液,即得到5μg/mg的链霉亲和素包被的磁微粒悬浮液。Take 0.1M PBS buffer solution (PH 7.4) and add it to a clean tube, add 0.1mg streptavidin to make the volume of the two 600μL; vortex mix for 5s; add 400μL 0.1M PBS buffer (pH7.4 dissolved 3M ammonium sulfate solution); vortex mix for 5s, and place it on a rolling mixer for rolling incubation at 37℃ for 2-4h. After incubation, place it on the magnetic stand for 1min until the magnetic bead solution becomes clear, and remove the supernatant; wash the magnetic beads once with 1000μL 100mM glycine solution (pH11.3): add 1000μL 100mM glycine solution (pH11.3), vortex for 5s, place it on the magnetic stand for 1min until the magnetic bead solution becomes clear, and remove the supernatant; wash the magnetic beads once with 1000μL 200mM glycine solution (pH2.8): add 1000μL 200mM glycine solution (pH2.8), vortex for 5s, place on the magnetic stand for 1min until the magnetic bead solution becomes clear, and remove the supernatant; 1000μL 50mM Tris-HCl (pH7.4 containing 140mM NaCl, 1% DB1130 and 0.1% Tween-20); wash the magnetic beads twice: First time: add 1000μL 50mM Tris-HCl (pH7.4 containing 140mM NaCl, 1% DB1130 and 0.1% Tween-20), place on a roller mixer and mix for 10min, then place on the magnetic stand for 1min until the magnetic bead solution becomes clear, and remove the supernatant; Second time: add 1000μL 50mM Tris-HCl (pH 7.4 containing 140 mM NaCl, 1% commercial blocking agent DB1130 and 0.1% Tween-20) was placed on a roller mixer and mixed overnight at 37°C, and then placed on a magnetic stand for 1 min until the magnetic bead solution became clear, and the supernatant was removed to obtain a 5 μg/mg streptavidin-coated magnetic particle suspension.
(2)碱性磷酸酶标记的鼠抗人IgE抗体的制备(2) Preparation of mouse anti-human IgE antibody labeled with alkaline phosphatase
将碱性磷酸酶标记的鼠抗人IgE用含1%鱼血清的MES缓冲液(pH 6.0)配制成0.3μg/mL的浓度,即得。The alkaline phosphatase-labeled mouse anti-human IgE was prepared at a concentration of 0.3 μg/mL using MES buffer (pH 6.0) containing 1% fish serum.
(3)校准品的制备(3) Preparation of calibrators
人血清用含1%鱼血清的Tris缓冲液(pH 7.4)进行稀释成0.0IU/mL、0.35IU/mL、0.70IU/mL、3.50IU/mL、17.50IU/mL、100.00IU/mL的梯度校准品,即得。Human serum was diluted with Tris buffer (pH 7.4) containing 1% fish serum to obtain a gradient calibrator of 0.0 IU/mL, 0.35 IU/mL, 0.70 IU/mL, 3.50 IU/mL, 17.50 IU/mL, and 100.00 IU/mL.
(4)生物素标记的鼠抗人IgE抗体的制备(4) Preparation of biotin-labeled mouse anti-human IgE antibody
取1.0mg鼠抗人IgE抗体,在0.1M PBS(PH 7.0)中室温下透析12h,中途换液1次,透析完成后,按照1:20的摩尔比加速10mM BNHS-Biotin避光反应30min。反应结束后,加入1%Tris进行封闭,再使用在0.1M PBS(PH 7.0)中室温下透析24h,中间换液一次,即得生物素标记的鼠抗人IgE抗体。Take 1.0 mg of mouse anti-human IgE antibody, dialyze it in 0.1M PBS (PH 7.0) at room temperature for 12 hours, change the liquid once in the middle, and after the dialysis is completed, accelerate the reaction of 10mM BNHS-Biotin in a molar ratio of 1:20 for 30 minutes in the dark. After the reaction is completed, add 1% Tris for blocking, and then dialyze it in 0.1M PBS (PH 7.0) at room temperature for 24 hours, change the liquid once in the middle, and obtain biotin-labeled mouse anti-human IgE antibody.
(5)生物素标记的牛奶组分过敏原的制备(5) Preparation of biotin-labeled milk component allergens
因4种蛋白等电点、分子量、氨基酸组成各不相同,故采用分别标记方式进行标记.。Since the four proteins have different isoelectric points, molecular weights, and amino acid compositions, they are labeled separately.
分别取1.0mg(α-乳清蛋白、β-乳球蛋白、牛血清白蛋白、酪蛋白、重组β-乳球蛋白),在0.1M PBS(PH 7.0)中室温下透析12h,中途换液1次,透析完成后,按照1:20的摩尔比加速10mM BNHS-Biotin避光反应30min。反应结束后,加入1%Tris进行封闭,再使用在0.1MPBS(PH 7.0)中室温下透析24h,中间换液一次,即得生物素标记α-乳清蛋白、生物素标记β-乳球蛋白、生物素标记牛血清白蛋白、生物素标记酪蛋白和生物素标记重组β-乳球蛋白。Take 1.0 mg (α-lactalbumin, β-lactoglobulin, bovine serum albumin, casein, recombinant β-lactoglobulin) respectively, dialyze in 0.1M PBS (PH 7.0) at room temperature for 12 hours, change the solution once in the middle, and accelerate the reaction of 10mM BNHS-Biotin in the dark for 30 minutes according to the molar ratio of 1:20. After the reaction is completed, add 1% Tris for blocking, and then dialyze in 0.1M PBS (PH 7.0) at room temperature for 24 hours, change the solution once in the middle, and obtain biotin-labeled α-lactalbumin, biotin-labeled β-lactoglobulin, biotin-labeled bovine serum albumin, biotin-labeled casein and biotin-labeled recombinant β-lactoglobulin.
实施例4:Embodiment 4:
采用磁微粒化学发光免疫分析间接法,检测人血清中牛奶组分过敏特异性的lgE抗体。The indirect method of magnetic particle chemiluminescent immunoassay was used to detect milk component allergy-specific IgE antibodies in human serum.
将待测样本与M磁微粒、R1生物素(Biotin)标记的牛奶组分过敏原混匀并在37℃孵育10min,经洗涤后加入R2碱性磷酸酶标记的鼠抗人lgE抗体反应10min,形成固相抗原-抗体-酶标二抗复合物,通过洗涤,未被结合的酶标抗体以及其它物质被去除。加入化学发光底物AMPPD,发光底物在碱性磷酸酶的催化下发射出光子,所产生的光子数与样本中的特异性lgE的浓度成正比。The sample to be tested is mixed with M magnetic particles and R1 biotin-labeled milk component allergens and incubated at 37°C for 10 minutes. After washing, R2 alkaline phosphatase-labeled mouse anti-human IgE antibody is added to react for 10 minutes to form a solid-phase antigen-antibody-enzyme-labeled secondary antibody complex. The unbound enzyme-labeled antibody and other substances are removed by washing. The chemiluminescent substrate AMPPD is added, and the luminescent substrate emits photons under the catalysis of alkaline phosphatase. The number of photons generated is proportional to the concentration of specific IgE in the sample.
试验例1:Test Example 1:
对本发明提供的牛奶组分过敏原特异性IgE检测试剂盒的线性、检测限准确性和精密度进行考察。The linearity, detection limit accuracy and precision of the milk component allergen-specific IgE detection kit provided by the present invention were investigated.
(1)线性(1) Linear
用该试剂盒检测不同浓度的标准品(其中每个浓度的标准品平行测试三次,取平均值),标准品的配制浓度与实际检测浓度用最小二乘法进行直线拟合,计算实测浓度与理论浓度的线性相关系数R,结果见表2:The kit was used to detect standards of different concentrations (each concentration of the standard was tested three times in parallel and the average value was taken). The prepared concentration of the standard and the actual detection concentration were fitted by the least squares method to calculate the linear correlation coefficient R between the measured concentration and the theoretical concentration. The results are shown in Table 2:
表2Table 2
本发明试剂盒的R≥0.99,符合临床评价要求。The R of the kit of the present invention is ≥0.99, which meets the clinical evaluation requirements.
(2)检测限(2) Detection limit
选择零浓度校准品(校准品稀释液),重复测定20次,得出20次测定结果的RLU值,计算其平均值M和标准差SD,得出(M+2SD)所对应的RLU值。测定相邻浓度校准品(主校准品S1)5次,记录其信号值,取平均值,根据零浓度校准品与相邻校准品之间的浓度-RLU值结果进行两点回归拟合得出一次方程,将(M+2SD)所对应的RLU值带入上述方程中,计算得出对应的浓度,即为最低检测限,结果见表3:Select a zero-concentration calibrator (calibrator dilution), repeat the measurement 20 times, obtain the RLU values of the 20 measurement results, calculate the average value M and standard deviation SD, and obtain the RLU value corresponding to (M+2SD). Measure the adjacent concentration calibrator (main calibrator S1) 5 times, record its signal value, take the average value, perform two-point regression fitting based on the concentration-RLU value results between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation, bring the RLU value corresponding to (M+2SD) into the above equation, and calculate the corresponding concentration, which is the minimum detection limit. The results are shown in Table 3:
表3Table 3
本发明试剂盒的检出限<0.1IU/mL,符合临床要求。The detection limit of the kit of the present invention is less than 0.1 IU/mL, which meets clinical requirements.
(3)准确度(3) Accuracy
将已知浓度的高值待测物A(Phadia定值)以不大于1:19的体积比加入低浓度血清B中得到待测样本,测试该样本,该待测样本的浓度应在试剂检测范围内,计算其回收率,结果见表4:A high-value analyte A (Phadia value) with a known concentration was added to a low-concentration serum B at a volume ratio of no more than 1:19 to obtain a test sample. The concentration of the test sample should be within the detection range of the reagent, and its recovery rate was calculated. The results are shown in Table 4:
表4Table 4
本发明试剂盒的检测结果的回收率在85%~115%,符合临床要求。The recovery rate of the detection result of the kit of the present invention is 85% to 115%, which meets the clinical requirements.
(4)精密度(4) Precision
用0.35IU/mL~0.70IU/mL和3.50IU/mL~17.50IU/mL两个浓度水平的样本各重复检测10次,计算测定结果的平均值和标准差,得到变异系数CV,结果见表5:The samples at two concentration levels of 0.35 IU/mL to 0.70 IU/mL and 3.50 IU/mL to 17.50 IU/mL were tested 10 times each, and the mean and standard deviation of the test results were calculated to obtain the coefficient of variation CV. The results are shown in Table 5:
表5Table 5
本实施例制备得到的试剂盒检测结果变异系数(CV)<10.0%,符合临床要求。The coefficient of variation (CV) of the test results of the kit prepared in this example is less than 10.0%, which meets clinical requirements.
(5)牛奶组分过敏样本检测结果,见表6所示:(5) Test results of milk component allergy samples are shown in Table 6:
表6Table 6
结果显示:与Phadia比较,本发明的牛奶组分检测试剂盒与Phadia的阴阳性符合率为100%,±1级符合率>85%,符合临床评价要求。该试剂盒可以同时快速检测被测者对牛奶4种组分过敏情况The results showed that compared with Phadia, the positive and negative consistency rate of the milk component detection kit of the present invention and Phadia was 100%, and the ±1 level consistency rate was >85%, which met the clinical evaluation requirements. The kit can simultaneously and quickly detect the subject's allergy to the four components of milk
试验例2:超声波处理对β-乳球蛋白与IgE结合的影响Experimental Example 2: Effect of Ultrasonic Treatment on the Binding of β-lactoglobulin and IgE
过敏原蛋白经过热处理、辐射、高压脉冲电场以及超声波等手段处理后,其分子量可能会发生改变。β-乳球蛋白单体由162个氨基酸组成,分子量为18.0kDa。The molecular weight of allergen proteins may change after being treated by heat treatment, radiation, high-voltage pulsed electric field, and ultrasound. The β-lactoglobulin monomer is composed of 162 amino acids and has a molecular weight of 18.0 kDa.
以超声波功率120W,分别以0min、10min、20min、30min、40min不同超声波处理时间,考察超声波处理时间对β-乳球蛋白分子量的影响,结果见图2所示。With an ultrasonic power of 120 W and different ultrasonic treatment times of 0 min, 10 min, 20 min, 30 min and 40 min, the effect of ultrasonic treatment time on the molecular weight of β-lactoglobulin was investigated. The results are shown in Figure 2.
从图2可以看出,根据标准蛋白电泳条带可以看出,与未处理的β-乳球蛋白相比,经不同超声波处理时间的β-乳球蛋白样品电泳条带无明显变化,说明超声波处理时间(120W,0-40min)不会改变β-乳球蛋白的分子量。As can be seen from Figure 2, according to the standard protein electrophoresis bands, compared with the untreated β-lactoglobulin, the electrophoresis bands of the β-lactoglobulin samples treated with different ultrasonic treatment times have no obvious changes, indicating that the ultrasonic treatment time (120W, 0-40min) will not change the molecular weight of β-lactoglobulin.
采用磁微粒化学发光间接法检测超声波处理时间对β-乳球蛋白与IgE结合能力的影响,实验结果如图3所示。The magnetic particle chemiluminescence indirect method was used to detect the effect of ultrasonic treatment time on the binding ability of β-lactoglobulin and IgE. The experimental results are shown in Figure 3.
从图中3可以看出,经不同超声波处理时间后,β-乳球蛋白的抗原性均有不程度的提升同程度的增加,最大达到12.05IU/mL,相比未处理样品增加了102.4%,但是与Phadia金标准比较,测值结果任显著性偏低。综上不同超声波时间β-乳球蛋白的IgE结合能力的影响不显著。因此,超声波处理对β-乳球蛋白的分子量的影响不大,可能是由于超声波处理没有影响到β-乳球蛋白肽段的完整性,但超声波处理能提升β-乳球蛋白与IgE的结合能力,可能是由于超声波诱导了β-乳球蛋白结构的该表,时期部分被掩盖的表位暴露出来,从而导致其与IgE结合能力上升,但当超声波时间过度时,超声波导致β-乳球蛋白的构象表位被破坏,而导致与IgE结合能力降低。但如图3所示,超声波处理最佳组合仍显著性偏低phadia结果,怀疑其为β-乳球蛋白的线性表位被掩盖。研究表明β-乳球蛋白的IgE过敏表位为AA41-60、AA106-125、AA149-162,这些过敏表位被90%~100%的牛乳过敏患者识别,因此我们对β-乳球蛋白进行重组。As can be seen from Figure 3, after different ultrasonic treatment times, the antigenicity of β-lactoglobulin has increased to varying degrees, with the maximum reaching 12.05IU/mL, an increase of 102.4% compared to the untreated sample, but compared with the Phadia gold standard, the measured value is still significantly lower. In summary, the effect of different ultrasonic treatment times on the IgE binding ability of β-lactoglobulin is not significant. Therefore, ultrasonic treatment has little effect on the molecular weight of β-lactoglobulin, which may be due to the fact that ultrasonic treatment does not affect the integrity of the β-lactoglobulin peptide segment, but ultrasonic treatment can enhance the binding ability of β-lactoglobulin with IgE, which may be due to the fact that ultrasound induces the structure of β-lactoglobulin, and the partially masked epitopes are exposed, thereby increasing its binding ability with IgE. However, when the ultrasonic time is excessive, ultrasound causes the conformational epitopes of β-lactoglobulin to be destroyed, resulting in a decrease in the binding ability with IgE. However, as shown in Figure 3, the best combination of ultrasonic treatment is still significantly lower than the Phadia result, and it is suspected that the linear epitopes of β-lactoglobulin are masked. Studies have shown that the IgE allergic epitopes of β-lactoglobulin are AA41-60, AA106-125, and AA149-162. These allergic epitopes are recognized by 90% to 100% of patients with cow milk allergy. Therefore, we recombined β-lactoglobulin.
试验例3:重组Bos d 5、天然Bos d 5、超声波处理及重组Bos d 5与天然Bos d 5联合超声波处理与IgE结合的比较分析Experimental Example 3: Comparative analysis of the binding of recombinant Bos d 5, natural Bos d 5, ultrasonic treatment, and recombinant Bos d 5 and natural Bos d 5 combined with ultrasonic treatment to IgE
对重组后的β-乳球蛋白进行SDS-PAGE检测如图4所示。The SDS-PAGE detection of the recombinant β-lactoglobulin is shown in FIG4 .
从图4可以看出,重组β-乳球蛋白分子量主要在18kDa附近,从图4中可观察到β-乳球蛋白条带的主要聚集在18kDa左右,说明重组β-乳球蛋白的分子量符合β-乳球蛋白的事实。As can be seen from Figure 4, the molecular weight of recombinant β-lactoglobulin is mainly around 18 kDa. From Figure 4, it can be observed that the β-lactoglobulin bands are mainly concentrated around 18 kDa, indicating that the molecular weight of recombinant β-lactoglobulin is consistent with the fact that β-lactoglobulin.
对天然β-乳球蛋白、重组β-乳球蛋白肽段、超声波处理天然β-乳球蛋白和将天然β-乳球蛋白、重组β-乳球蛋白肽段进行超声波处理与IgE结合能力的分析,得到结果如图5所示。The results of the analysis of the IgE binding ability of natural β-lactoglobulin, recombinant β-lactoglobulin peptides, ultrasonically treated natural β-lactoglobulin, and ultrasonically treated natural β-lactoglobulin and recombinant β-lactoglobulin peptides are shown in FIG5 .
从图5可以看出,重组β-乳球蛋白具有与IgE结合能力,说明重组的β-乳球蛋白肽段显示了与IgE结合的线性表位,但其与IgE结合能力显著性低于天然β-乳球蛋白,是由于重组β-乳球蛋白仅是一条肽段,不具有β-乳球蛋白天然的α螺旋、β折叠等二级结构,且据前人研究结果得出,β-乳球蛋白的抗原性大部分是由蛋白质的二级结构决定。结果显示重组蛋白与血清IgE反应的明显低于天然牛奶过提取物β-乳球蛋白,这是由于重组牛奶β-乳球蛋白只存在线性表位。通过超声处理天然β-乳球蛋白和重组β-乳球蛋白与血清IgE检测结果,混合组的IgE检测含量为16.29IU/mL与Phadia测试结果无显著性区别,因此,在检测β-乳球蛋白疾病时,采用超声波天然提取β-乳球蛋白和重组β-乳球蛋白的混合物更有优势。As can be seen from Figure 5, recombinant β-lactoglobulin has the ability to bind to IgE, indicating that the recombinant β-lactoglobulin peptide segment shows a linear epitope that binds to IgE, but its ability to bind to IgE is significantly lower than that of natural β-lactoglobulin. This is because recombinant β-lactoglobulin is only a peptide segment and does not have the secondary structures of α-helix, β-folding, etc. of natural β-lactoglobulin. According to previous research results, the antigenicity of β-lactoglobulin is mostly determined by the secondary structure of the protein. The results show that the recombinant protein reacts significantly less with serum IgE than natural milk extract β-lactoglobulin, which is due to the presence of only linear epitopes in recombinant milk β-lactoglobulin. The results of ultrasonic treatment of natural β-lactoglobulin and recombinant β-lactoglobulin and serum IgE test showed that the IgE detection content of the mixed group was 16.29IU/mL, which was not significantly different from the Phadia test results. Therefore, when detecting β-lactoglobulin diseases, the mixture of ultrasonic natural extract β-lactoglobulin and recombinant β-lactoglobulin is more advantageous.
试验例4:α-亚麻酸诱导α-乳白蛋白与IgE结合的影响Experimental Example 4: Effect of α-linolenic acid on the binding of α-lactalbumin to IgE
利用非还原性SDS-PAGE鉴定了α-亚麻酸与α-乳白蛋白形成复合物的蛋白分子量的变化,如图6所示。The change in the molecular weight of the protein complex formed by α-linolenic acid and α-lactalbumin was identified by non-reducing SDS-PAGE, as shown in FIG6 .
从图6的电泳图可观察到,α-亚麻酸对α-乳白蛋白的分子量有不同程度的影响。与未处理的α-乳白蛋白相比,经α-亚麻酸(分子量278.43)处理后的蛋白分子量出现轻微的变化,主要是蛋白条带发生了轻微的下移。It can be observed from the electrophoresis diagram of Figure 6 that α-linolenic acid has different degrees of influence on the molecular weight of α-lactalbumin. Compared with untreated α-lactalbumin, the molecular weight of the protein treated with α-linolenic acid (molecular weight 278.43) changed slightly, mainly because the protein band shifted slightly downward.
通过磁微粒化学发光法评估了α-亚麻酸处理后α-乳白蛋白的抗原性(IgE结合能力)的变化,结果见图7所示。The changes in the antigenicity (IgE binding ability) of α-lactalbumin after treatment with α-linolenic acid were evaluated by magnetic particle chemiluminescence method. The results are shown in FIG7 .
如图7显示了α-亚麻酸处理后α-乳白蛋白的IgE结合能力的变化,在三个不同等级的α-乳白蛋白的过敏血清中,α-亚麻酸处理后α-乳白蛋白的IgE结合能力均显著性增强,且与Phadia金标准比较,对照组的测值偏低,经α-亚麻酸处理后α-乳白蛋白的IgE结合能力无明显区别。Figure 7 shows the changes in the IgE binding capacity of α-lactalbumin after treatment with α-linolenic acid. In the three different levels of α-lactalbumin allergic sera, the IgE binding capacity of α-lactalbumin was significantly enhanced after treatment with α-linolenic acid, and compared with the Phadia gold standard, the measured values of the control group were lower, and there was no significant difference in the IgE binding capacity of α-lactalbumin after treatment with α-linolenic acid.
IgE结合能力的增加主要是因为α-亚麻酸处理促使α-乳白蛋白的空间结构不断展开,致使蛋白内部掩埋的IgE表位暴露出来,导致其IgE结合能力的增加。此外,α-亚麻酸处理促使α-乳白蛋白IgE结合能力的增加可能是由α-亚麻酸处理促使α-乳白蛋白使其逐渐暴露的线性表位引起的。The increase in IgE binding capacity is mainly due to the fact that α-linolenic acid treatment causes the spatial structure of α-lactalbumin to unfold continuously, exposing the IgE epitopes buried inside the protein, leading to an increase in its IgE binding capacity. In addition, the increase in IgE binding capacity of α-lactalbumin caused by α-linolenic acid treatment may be caused by the linear epitopes that α-lactalbumin gradually exposes due to α-linolenic acid treatment.
试验例5:考察封闭剂对性能的影响Test Example 5: Investigating the effect of sealant on performance
采用BSA和商业阻断剂DB1130封闭裸磁珠后,磁珠非特异性吸附样本结果见图8所示,不同封闭剂测试临床样本结果见图9所示。After the naked magnetic beads were blocked with BSA and the commercial blocking agent DB1130, the results of nonspecific adsorption of samples by magnetic beads are shown in Figure 8 , and the results of clinical samples tested with different blocking agents are shown in Figure 9 .
从图8可以卡出,在降低磁珠非特异性吸附样本上的作用,采用BSA和商业阻断剂DB1130封闭裸磁珠是一致的,磁珠未进行封闭时出现了明显的吸附,导致测试光子数显著性高于BSA和商业阻断剂DB1130。As can be seen from Figure 8, the effect of blocking bare magnetic beads with BSA and the commercial blocker DB1130 on reducing nonspecific adsorption of samples is consistent. When the magnetic beads are not blocked, obvious adsorption occurs, resulting in a significantly higher test photon number than BSA and the commercial blocker DB1130.
但采用BSA封闭磁珠会导致如图9结果,在检测过程中,BSA可能与样本中的IgE结合,从而与二抗形成复合物,从而导致测试结果假阳。同时由于BSA属于牛源性蛋白质分子量较大,在测试会占据抗原与抗体结合的位置,形成空间位阻,从而导致漏检,但采用化学合成的商业阻断剂DB1130可以避免由于BSA引起的漏检和假阳。However, using BSA to block magnetic beads will result in the results shown in Figure 9. During the test, BSA may bind to IgE in the sample, thereby forming a complex with the secondary antibody, resulting in a false positive test result. At the same time, since BSA is a bovine protein with a large molecular weight, it will occupy the position where the antigen and antibody bind, forming a steric hindrance, resulting in missed detection. However, the use of chemically synthesized commercial blocking agent DB1130 can avoid missed detection and false positives caused by BSA.
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
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