A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2–4 and Cκ Domains Is an Alternative Reagent to Human IgE

Abstract

Abstract 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 (Cγ1-Cε2–4, each ∼53 kDa) and two constant κ L chains (Cκ, each ∼12 kDa) and lacks a V domain. The presence of Cγ1 instead of Cε1 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.