Serum-free hybridoma production of anti-ovalbumin IgE monoclonal antibodies: a practical approach for milligram-scale yield

Monoclonal immunoglobulin E (IgE) antibodies are essential tools for investigating allergen-specific immune responses; however, their production remains technically challenging due to the low frequency and instability of IgE-secreting B cells. In addition, reliance on serum-containing culture systems limits reproducibility and scalability of hybridoma-based antibody purification.In this study, we aimed to develop a reproducible hybridoma-based workflow for the production of anti-ovalbumin (OVA) IgE monoclonal antibodies under serum-free conditions. BALB/c mice were sensitized with OVA via subcutaneous and intraperitoneal routes, with or without aluminum hydroxide (alum) as an adjuvant. Serum total IgE and OVA-specific IgE levels were assessed to evaluate sensitization efficiency. Splenocytes from high-responder mice were fused with myeloma cells to generate IgE-secreting hybridomas, which were subsequently sub-cloned and adapted to serum-free medium through a stepwise reduction strategy. Antibody production was carried out at the laboratory scale, followed by ammonium sulfate concentration and structural validation by SDS-PAGE.Subcutaneous sensitization induced stronger IgE responses compared to the intraperitoneal route, while alum significantly enhanced OVA-specific IgE production. Hybridoma generation yielded viable colonies with a fusion efficiency of 19.2%, from which stable IgE-secreting clones were obtained. Successful adaptation to serum-free medium was achieved with maintained cell viability and sustained antibody production over a 14-day culture period. The final OVA-specific IgE concentration reached approximately 4 mg/ml, with an 80% recovery following ammonium sulfate precipitation. SDS-PAGE analysis confirmed the expected molecular structure of IgE under reducing and non-reducing conditions.These findings demonstrate a practical and reproducible approach for generating OVA-specific IgE monoclonal antibodies at laboratory scale under serum-free conditions, providing a useful platform for allergy research and immunological applications.