Generation of Antibodies Selectively Recognizing Epitopes in a Formaldehyde-Fixed Cell-Surface Antigen Using Virus-like Particle Display and Hybridoma Technology

verfasst von

Stefanie Schatz, Lena Willnow, Monika Winkels, Jamila Franca Rosengarten, Benjamin Theek, Ian C.D. Johnston, Jörn Stitz

Abstract

Efficient induction of target-specific antibodies can be elicited upon immunization with highly immunogenic virus-like particles (VLPs) decorated with desired membrane-anchored target antigens (Ags). However, for example, for diagnostic purposes, monoclonal antibodies (mAbs) are required to enable the histological examination of formaldehyde-fixed paraffin-embedded (FFPE) biopsy tissue samples. Aiming at the generation of FFPE-antigen-specific mAbs and as a proof of concept (POC), we first established a simplified protocol using only formaldehyde and 90 °C heat fixation (FF90) of cells expressing the target Ag nerve growth factor receptor (NGFR). The FF90 procedure was validated using flow cytometric analysis and two mAbs recognizing either the native and FFPE-Ag or exclusively the native Ag. C-terminally truncated NGFR (trNGFR)-displaying native and FF90-treated VLPs derived from HIV-1 did not reveal distinctive changes in particle morphology using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. Mice were subsequently repetitively immunized with trNGFR-decorated FF90-VLPs and hybridoma technology was used to establish mAb-producing cell clones. In multiple screening rounds, nine cell clones were identified producing mAbs distinctively recognizing epitopes in FF90- and FFPE-NGFR. This POC of a new methodology should foster the future generation of mAbs selectively targeting FFPE-fixed cell-surface Ags.

Organisationseinheit(en)

Institut für Technische Chemie

Externe Organisation(en)

Technische Hochschule Köln
Miltenyi Biotec GmbH

Typ

Artikel

Journal

Antibodies

Band

12

Anzahl der Seiten

20

ISSN

2073-4468

Publikationsdatum

05.09.2023

Publikationsstatus

Veröffentlicht

Peer-reviewed

Ja

ASJC Scopus Sachgebiete

Immunologie und Allergologie, Immunologie, Wirkstoffforschung

Ziele für nachhaltige Entwicklung

SDG 3 – Gute Gesundheit und Wohlergehen

Elektronische Version(en)

https://doi.org/10.3390/antib12030057 (Zugang: Offen)