Evaluating IgE TRAP Fc Function

To evaluate whether IgD/IgG4 hybrid Fc domain of IgE_TRAP lacks IgG Fc-mediated function unlike omalizumab, ELISA plates were coated with Nitrophenyl-hapten (NP)-BSA (Bioresearch technologies, UK; 10 μg mL⁻¹) at 4 °C overnight and, after washing with PBS, incubated with anti-NP chimeric human IgE (JW8/1; GeneTax, USA; #GTX17414; 1:3 dilution) for 1 h at 37 °C. After washing with IMDM media containing 20% FBS, the plates were incubated with various concentrations of each drug (IgE_TRAP, IgE_TRAP-IgG1M2 or omalizumab) for 1 h at 37 °C. IgE_TRAP-IgG1M2, which is IgE_TRAP with superior ability to induce IgG1 Fc-mediated side effects, was used as a positive control. The plates were cultured with NK103 cells (1 × 10⁵ cells) expressing FcγRIIIA for 5 h at 37 °C in CO₂ incubator following a washing step with IMDM media containing 20% FBS. During the culture, NK cells were activated through FcγRIIIA to secrete granzyme B, which was used as an indicator of IgG-mediated function. Granzyme B in culture supernatant was measured with human granzyme B DuoSet ELISA kit (R&D systems, USA) according to the manufacturer’s instruction. NK103 cells are FcγRIIIA-expressing NK cell line derived from human lymphoma, NK101^{46 (link)}. NK103 was kindly provided by Dr. Sae Won Kim (SL-GIBEN Inc., Korea).