Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
Clin Exp Allergy. 2011 Feb;41(2):270-80. doi: 10.1111/j.1365-2222.2010.03666.x. Epub 2010 Dec 10.
Allergen recognition by IgE antibodies is a key event in allergic inflammation.
To construct a plasmid for the expression of human monoclonal IgE antibodies of any desired specificity and to express IgE specific for the major timothy grass pollen allergen Phl p 5.
In a first step, the DNA sequence coding for the IgG(1) heavy chain was excised and replaced by the sequence coding for the human ɛ constant region gene in plasmid pLNOH2 expressing a human Phl p 5-specific IgG(1) heavy chain. Then, this construct together with a second plasmid expressing the corresponding Phl p 5-specific light chain was co-expressed in COS-7 cells. The Phl p 5-specific IgE (rhuMabEP5) was analysed for allergen-specificity and isotype by ELISA. Cross-reactivity of rhuMabEP5 was investigated by immunoblotting using pollen extracts from various grass species. The allergenic activity of Phl p 5 was studied by exposing rat basophil leukaemia (RBL) cells expressing human-FcɛRI to rhuMabEP5 and Phl p 5.
We report the construction of vector pLNOH2-P5IgE, for the expression of human IgE and exemplify its usefulness by the production of a complete and functional human monoclonal IgE (rhuMabEP5). rhuMabEP5 is specific for the grass pollen allergen Phl p 5 and cross-reacts with group 5 allergens in natural grass pollen extracts. RBL-release assays with rhuMabEP5 demonstrated that oligomerization does not contribute to the high allergenic activity of Phl p 5.
Plasmid pLNOH2-P5IgE allowed the production of a fully functional human monoclonal IgE antibody specific for Phl p 5. Recombinant human IgE antibodies of defined specificity represent useful tools to investigate mechanisms underlying IgE-mediated allergies.
IgE 抗体对过敏原的识别是过敏炎症的关键事件。
构建表达任何所需特异性的人单克隆 IgE 抗体的质粒，并表达针对主要豚草花粉过敏原 Phl p 5 的 IgE。
在第一步中，从质粒 pLNOH2 中切除编码 IgG(1)重链的 DNA 序列，并用人 ε 恒定区基因序列取代，该质粒表达针对人 Phl p 5 特异性 IgG(1)重链。然后，将该构建体与表达相应 Phl p 5 特异性轻链的第二个质粒共表达在 COS-7 细胞中。通过 ELISA 分析 Phl p 5 特异性 IgE（rhuMabEP5）的过敏原特异性和同种型。使用来自各种草种的花粉提取物通过免疫印迹研究 rhuMabEP5 的交叉反应性。通过暴露表达人-FcɛRI 的大鼠嗜碱性白血病（RBL）细胞来研究 Phl p 5 的过敏原活性 rhuMabEP5 和 Phl p 5。
我们报告了表达人 IgE 的载体 pLNOH2-P5IgE 的构建，并通过生产完整且功能齐全的人单克隆 IgE（rhuMabEP5）来举例说明其用途。rhuMabEP5 特异性针对草花粉过敏原 Phl p 5 ，并与天然草花粉提取物中的 5 组过敏原发生交叉反应。用 rhuMabEP5 进行 RBL 释放测定表明，寡聚化对 Phl p 5 的高过敏原活性没有贡献。
质粒 pLNOH2-P5IgE 允许生产针对 Phl p 5 的完全功能的人单克隆 IgE 抗体。具有明确特异性的重组人 IgE 抗体是研究 IgE 介导的过敏反应机制的有用工具。
