Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression

Schatz, Stefanie; van Dijk, Femke Harmina; Dubiel, Aleksandra Elzbieta; Cantz, Tobias; Eggenschwiler, Reto; Stitz, Jörn

doi:10.1007/978-1-0716-3279-6_20

Cited by 1 publication

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“…Subsequently, CFSNs of VLP producer cell pools were normalized to a concentration of 45 ng/mL p24-Gag and NGFR concentrations were investigated by conducting an NGFR-specific ELISA. Although parental 293-F cells expressed wild-type NGFR [27], no NGFR was detectable in the CFSN of 293-F cells (Table 1). Thus, wild-type full-length NGFR or its degradation products were not secreted in notable amounts (Table 1).…”

Section: Characterization Of Vlp Producer Cell Poolsmentioning

confidence: 99%

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

Schatz,

Willnow,

Winkels

et al. 2023

Antibodies

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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.

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Section: Characterization Of Vlp Producer Cell Poolsmentioning

confidence: 99%