目的:通过噬菌体展示技术制备抗人IgE纳米抗体,将其用于磁微粒化学发光平台建立一种猫皮屑特异性IgE抗体超敏检测方法.方法:在WHO/IUIS过敏原数据库中检索猫致敏蛋白信息,合成序列后在原核表达系统中表达和纯化猫皮屑致敏蛋白Fel d 1.使用人源IgE免疫新疆单峰驼提取淋巴细胞中的RNA建立噬菌体展示纳米抗体库,分析库容、多样性和插入率,通过筛选和鉴定获得抗人IgE纳米抗体.使用重组过敏原偶联磁微粒和吖啶酯标记纳米抗体,建立磁微粒化学法猫皮屑特异性IgE抗体检测方法.结果:噬菌体展示库的库容为1.88×108 cfu/ml,插入率为93.6%,纳米抗体纯度>95%.检测方法的线性范围为0.1～100 U/ml,与ImmunoCAP 检测系统临床对比有较好的一致性.结论:成功建立了基于纳米抗体的猫皮屑特异性IgE抗体超敏检测方法,为猫过敏性疾病辅助诊断提供了技术基础.
Establishment and performance evaluation of nanobody-based cat dander specific IgE antibody hypersensitivity assay
Objective:To prepare anti-human IgE nanobody by phage display technology,and to establish a method for hyper-sensitivity detection of cat dander specific IgE antibody.Methods:Allergen bio-information of cat was searched in WHO/IUIS Allergen Database.After synthesizing sequence,recombinant cat dander allergenic protein Fel d 1 was expressed and purified in prokaryotic ex-pression system.Human IgE was used to immunize Bactrian camel and RNA were extracted from lymphocyte to construct phage dis-play library.Library capacity,diversity and insertion rate were analyzed,anti-human IgE nanobody were obtained by screening and identification.A magnetic particle chemical method for cat dander specific IgE antibody detection was established using recombinant allergen-coupled magnetic particles and acridine ester-labeled nanobodies.Results:Capacity of phage display library was 1.88×108 cfu/ml,insertion rate was 93.6%,and purity of nanobody was>95%.Linear range of the method based on nanobody was 0.1～100 U/ml,who was consistent with ImmunoCAP detection system by clinical data.Conclusion:Nanobody-based cat dander specific IgE antibody hypersensitivity assay is successfully prepared,providing a technical basis for auxiliary diagnosis of cat allergic diseases.
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