In this study, a novel recombinant antibody fragment IgCw-γ1ε2-4/κ (IgCw-γε/κ) was constructed and evaluated for its potential as an alternative isotype control, which can be used in place of IgE. IgCw-γε/κ (~140 kDa) is composed of two hybrid human constant heavy chains (Cγ1-hinge region-Cε2-4) and two human constant kappa light chains (Cκ). IgCw-γε/κ was expressed as an assembled form of the heavy and light chains, and the production yield of IgCw-γε/κ obtained from a culture of transfected HEK293F cells was 17 mg/L, which is comparable to that of full-size IgE. Binding of IgCw-γε/κ to human Fc epsilon receptor Ⅰ (FcεRⅠ) expressed on RBL-2H3-hFcεRⅠα cells was confirmed using flow cytometry. A standard curve generated using IgCw-γε/κ was used for the measurement of IgE concentrations. Further, the sensitization with IgCw-γε/κ of RBL-2H3-hFcεRⅠα cells and subsequent stimulation with anti-human kappa light chain antibody induced β-hexosaminidase release. IgCw-γε/κ also acted as an FcεRⅠ blocker to suppress IgE-FcεRⅠ interaction. Taken together, these results demonstrate that IgCw-γε/κ can be used as a functional alternative isotype control, instead of full-size IgE, in biomedical research fields.
