Production and characterization of enzymatically generated immunoglobulin E (IgE) fragments of anti-ovalbumin IgE monoclonal antibody in mice

Most of our knowledge of allergen-specific immunoglobulin E (IgE) antibody molecules has come from studies using IgE found in patient serum. However, the ability to study the fine molecular details of the antibody-allergen interaction is limited due to the polyclonal nature and exceedingly low concentration of IgE in human serum.Advancements in hybridoma technologies allow for the generation and molecular definition of naturally occurring allergen-specific IgE monoclonal antibodies. Thus, we aimed in this study to produce anti-ovalbumin (OVA)IgE monoclonal antibodies and characterize the proteolytic cleavage fragments of IgEin BALB/c mice.,OVA was administered subcutaneously and intraperitoneally to twenty mice, either with or without an aluminum hydroxide (alum)adjuvant.Serum total IgE and anti-OVA IgE levels were assessed to evaluate sensitization efficacy and to compare the influence of administration route and adjuvant.Myeloma cells and splenocytes from the highest IgE responders were fused to generate hybridomas that secreted anti-OVA monoclonal IgE. The purified antibody was subjected to controlled enzymatic digestion using papain, pepsin, and trypsin. The resulting fragments were analyzed using SDS-PAGE to determine their molecular characteristics.,The present results revealed that subcutaneous immunization was superior to intraperitoneal immunization for eliciting strong anti-OVA IgE response, and that alum significantly amplified this effect. In addition, SDS-PAGE analysis for papain cleavage of anti-OVA IgE yielded two major IgE fragments of approximately 80 kDa and 50 kDa.The current findings may highlight the feasibility of producing monoclonal IgE by conventional PEG-mediated hybridoma technology and characterizing monoclonal IgE fragments derived from enzymatic cleavage,K