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

Prema S. Prakash, Nafal J. S. Barwary, Michael H. W. Weber, Daniel Wan, Ivan Conejeros, Bernardo Pereira Moreira, Waleed S. Alharbi, Jaap J. van Hellemond, Jude Akinwale, Franco H. Falcone*

*Corresponding author for this work

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Abstract

Rat basophilic leukaemia (RBL) cells have been used for decades as a model of high-affinity Immunoglobulin E (IgE) receptor (FcεRI) 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 FcεRIα cDNA used for transfection, which resulted in the removal of known endoplasmic reticulum retention signals and high surface expression of human FcεRIα* in huNPY-mRFP cells (where * denotes the penta-substituted variant), comparable to the ~500,000 FcεRIα molecules per cell in the RS-ATL8 humanised luciferase reporter, which is a human FcεRIα/FcεRIγ 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-1β and, to a lesser extent, by EgEF-1δ, which had been previously described as IgE-immunoreactive EgEF-1β/δ.

Original languageEnglish
Article number2063
Number of pages13
JournalDiagnostics
Volume12
Issue number9
DOIs
Publication statusPublished - Sep 2022

Bibliographical note

Funding Information:
This work was funded by the LOEWE Centre DRUID within the Hessian Excellence Initiative to FF.

Publisher Copyright:
© 2022 by the authors.

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