Department of Microbiology, Ajou University School of Medicine, Suwon, South Korea.
Department of Biomedical Sciences, Graduate School, Ajou University, Suwon, South Korea; and.
J Immunol. 2022 Feb 1;208(3):772-779. doi: 10.4049/jimmunol.2100576. Epub 2022 Jan 12.
Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ-Cε, each ∼53 kDa) and two constant κ L chains (C, each ∼12 kDa) and lacks a V domain. The presence of Cγ instead of Cε within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of ∼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A β-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays.
人 IgE 可用于免疫学检测，例如 FcεRI 阳性细胞的致敏和 IgE 测量。在本研究中，我们报告了一种重组 Ig 片段 IgCw-γεκ 的开发，作为人 IgE 的替代试剂。IgCw-γεκ（130 kDa）由两个杂交恒定 H 链区（Cγ-Cε，每个53 kDa）和两个恒定 κ L 链（C，每个~12 kDa）组成，并且缺乏 V 结构域。H 链内的 Cγ而不是 Cε的存在增加了产量，并促进了 H 和 L 链的组装。IgCw-γεκ在培养的人胚肾 293F 细胞中产生，产量约为 27 mg/L。IgCw-γεκ与在大鼠嗜碱性白血病-2H3 细胞表面表达的人 FcεRIαRs 结合。β-半乳糖苷酶释放测定显示 IgCw-γεκ 的生物学活性与 IgE 相当。使用 IgCw-γεκ 作为标准测量的 IgE 浓度与使用 IgE 作为标准测量的浓度相似。这些结果表明，IgCw-γεκ 分子保留了 IgE 的基本特征，但不与 Ag 发生交叉反应，使其成为各种免疫学检测中使用的 IgE 同种型参考的替代物。
