The Humanised NPY-mRFP RBL Reporter Cell Line Is a Fast and Inexpensive Tool for Detection of Allergen-Specific IgE in Human Sera

Rat basophilic leukaemia (RBL) cells have been used for decades as a model of high-affinity Immunoglobulin E (IgE) receptor (Fc epsilon RI) signalling. Here, we describe the generation and use of huNPY-mRFP, a new humanised fluorescent IgE reporter cell line. Fusion of Neuropeptide Y (NPY) with monomeric red fluorescent protein (mRFP) results in targeting of fluorescence to the granules and its fast release into the supernatant upon IgE-dependent stimulation. Following overnight sensitisation with serum, optimal release of fluorescence upon dose-dependent stimulation with allergen-containing extracts could be measured after 45 min, without cell lysis or addition of any reagents. Five substitutions (D194A, K212A, K216A, K226A, and K230A) were introduced into the Fc epsilon RI alpha cDNA used for transfection, which resulted in the removal of known endoplasmic reticulum retention signals and high surface expression of human Fc epsilon RI alpha* in huNPY-mRFP cells (where * denotes the penta-substituted variant), comparable to the similar to 500,000 Fc epsilon RI alpha molecules per cell in the RS-ATL8 humanised luciferase reporter, which is a human Fc epsilon RI alpha/Fc epsilon RI gamma double transfectant. The huNPY-mRFP reporter was used to demonstrate engagement of specific IgE in sera of Echinococcus granulosus infected individuals by E. granulosus elongation factor EgEF-1 beta and, to a lesser extent, by EgEF-1 delta, which had been previously described as IgE-immunoreactive EgEF-1 beta/delta.