Enhanced sensitivity of capture IgE-ELISA based on a recombinant Der f 1/2 fusion protein for the detection of IgE antibodies targeting house dust mite allergens
Enhanced sensitivity of capture IgE-ELISA based on a recombinant Der f 1/2 fusion protein for the detection of IgE antibodies targeting house dust mite allergens
复制标题
基于重组 Der f 1/2 融合蛋白的捕获 IgE-ELISA 的灵敏度增强，用于检测针对屋尘螨过敏原的 IgE 抗体
DOI：
10.3892/mmr.2019.10050
复制
发表时间：
2019-05-01
影响因子：
3.4
通讯作者：
Ji, Kunmei
中科院分区：
文献类型：
--
作者：
Zhang, Zhen;Cai, Zelang;Ji, Kunmei
过敏原特异性免疫球蛋白（IG）E的检测是诊断IgE介导的过敏性疾病的重要方法。间接IgE-ELISA方法针对过敏原提取物的灵敏度受到高IgG滴度和提取物中有效过敏原成分含量低的干扰的限制。为了克服这些局限性，开发了一种基于重组Der f 1/Der f 2融合蛋白（rDer f 1/2）的新型捕获IgE-ELISA，以提高对结合来自屋尘螨（HDM）物种粉尘螨的过敏原的IgE的灵敏度。构建pET 28-Der f 1/2，并在大肠杆菌BL 21（DE 3）pLysS中表达。用免疫印迹法、斑点印迹法和间接免疫酶联免疫吸附试验（ELISA）对纯化的融合蛋白进行鉴定。通过用奥马珠单抗包被威尔斯孔并与血清、生物素化的Der f 1/2、辣根过氧化物酶缀合的链霉亲和素和3，3，5，5-四甲基联苯胺连续孵育来进行捕获-ELISA。间接ELISA和捕获ELISA的HDM过敏原特异性IgE结合的相对灵敏度进行了测定，从非过敏个体的血清作为对照组。rDer f 1/2以包涵体的形式表达，包涵体包含重折叠的蛋白，然后纯化。与HDM过敏血清的结合率（24/28，85.8%）高于rDer f 1（21/28，75.0%）或rDer f 2（22/28，78.6%）。在随机抽取的71例HDM过敏患者血清中，捕获ELISA法检测HDM特异性IgE的灵敏度（71/71，100%）高于间接ELISA法（68/71，95.8%）（P
The detection of allergen-specific immunoglobulin (Ig)E is an important method for the diagnosis of IgE-mediated allergic diseases. The sensitivity of the indirect IgE-ELISA method against allergen extracts is limited by interference from high IgG titers and low quantities of effectual allergen components in extracts. To overcome these limitations, a novel capture IgE-ELISA based on a recombinant Der f 1/Der f 2 fusion protein (rDer f 1/2) was developed to enhance the sensitivity to IgEs that bind allergens from the house dust mite (HDM) species Dermatophagoides farina. pET28-Der f 1/2 was constructed and expressed in Escherichia coli BL21 (DE3) pLysS. The purified fusion protein was evaluated by IgE western blotting, IgE dot blotting and indirect IgE-ELISA. Capture-ELISA was performed by coating wells with omalizumab and incubating in series with sera, biotinylated Der f 1/2, horseradish peroxidase-conjugated streptavidin and 3,3,5,5-tetramethylbenzidine. The relative sensitivities of indirect-ELISA and capture-ELISA for HDM allergen-specific IgE binding were determined; sera from non-allergic individuals were used as the control group. rDer f 1/2 was expressed in the form of inclusion bodies comprising refolded protein, which were then purified. It exhibited increased IgE-specific binding (24/28, 85.8%) than rDer f 1 (21/28, 75.0%) or rDer f 2 (22/28, 78.6%) with HDM-allergic sera. Furthermore, in a random sample of HDM-allergic sera (n=71), capture-ELISA (71/71, 100%) was more sensitive than indirect-ELISA (68/71, 95.8%) for the detection of HDM-specific IgEs (P