Details
| Original language | English |
|---|---|
| Article number | 57 |
| Number of pages | 20 |
| Journal | Antibodies |
| Volume | 12 |
| Issue number | 3 |
| Publication status | Published - 5 Sept 2023 |
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.
Keywords
- antibody discovery, antigen display, cell-surface antigen, formaldehyde-fixed paraffin-embedded (FFPE) tissue samples, hybridoma technology, nerve growth factor receptor, virus-like particles
ASJC Scopus subject areas
- Medicine(all)
- Immunology and Allergy
- Immunology and Microbiology(all)
- Immunology
- Pharmacology, Toxicology and Pharmaceutics(all)
- Drug Discovery
Sustainable Development Goals
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: Antibodies, Vol. 12, No. 3, 57, 05.09.2023.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Generation of Antibodies Selectively Recognizing Epitopes in a Formaldehyde-Fixed Cell-Surface Antigen Using Virus-like Particle Display and Hybridoma Technology
AU - Schatz, Stefanie
AU - Willnow, Lena
AU - Winkels, Monika
AU - Rosengarten, Jamila Franca
AU - Theek, Benjamin
AU - Johnston, Ian C.D.
AU - Stitz, Jörn
N1 - Funding Information: This research was funded by the German Federal Ministry of Education and Research, funding program Forschung an Fachhochschulen, grant numbers 13FH242PX6 and 13FH767IA6 to J.S.
PY - 2023/9/5
Y1 - 2023/9/5
N2 - 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.
AB - 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.
KW - antibody discovery
KW - antigen display
KW - cell-surface antigen
KW - formaldehyde-fixed paraffin-embedded (FFPE) tissue samples
KW - hybridoma technology
KW - nerve growth factor receptor
KW - virus-like particles
UR - http://www.scopus.com/inward/record.url?scp=85172098864&partnerID=8YFLogxK
U2 - 10.3390/antib12030057
DO - 10.3390/antib12030057
M3 - Article
AN - SCOPUS:85172098864
VL - 12
JO - Antibodies
JF - Antibodies
SN - 2073-4468
IS - 3
M1 - 57
ER -