A robust and stable reporter gene bioassay for anti-IgE antibodies.

Immunoglobin E (IgE)-related allergy constitutes a high proportion in allergic diseases. The production of specific IgE is key to evoking serial cascades and pathological reactions. Thus, targeting IgE is a different therapeutic approach from symptomatic treatments. Monoclonal antibodies (mAbs) against IgE were developed and a humanized antibody, omalizumab, was approved by five countries. It could inhibit the binding of IgE with epsilon receptor I of crystallizable fragment (FcεRI), thus preventing anaphylactic reactions. However, no bioactivity assay, which is the critical quality attribute and should thoroughly reflect the clinical mechanism, has been established to date. In commercial lot release, only the enzyme-linked immunosorbent assay (ELISA) method was applied, which only reflects the binding of omalizumab to IgE but not the subsequent reaction. In scientific research works, human FcεRI-transfected RBL-2H3 cells were used to indicate degranulation based on the detection of ß-hexosaminidase. Nevertheless, this method needs much work to stabilize the response and, hence, is not suitable for routine usage in commercial production and control of antibodies. To evaluate the bioactivity of anti-IgE antibodies including omalizumab using a simple assay that reflects the following mechanism of actions (MOA) after binding, we established an RBL-2H3 cell line transfected with both the α subunit of human FcεRI and nuclear factor-activated T cell (NFAT) response elements, the latter is conjugated with a luciferase gene, which could shed luminescence when substrates exist. The method was proven to possess good specificity, accuracy, linearity, and precision and may be utilized as a supplement to anti-IgE antibody bioactivity assays in terms of development, lot release, stability, and comparability studies. Graphical abstract The mechanism sketch of reporter gene assay for bioactivity determination of anti-IgE antibodies by RBL-2H3/FcεRIα/NFAT-Luc cells (left) and representative curves generated by the reporter gene assay (right).