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

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.

CD133 expression is associated with small round blue cell tumour morphology in human central nervous system neoplasms.

AIMS: CD133 is considered to be a marker for brain tumour-initiating cells. However, most data on CD133 are derived from animal or in-vitro studies. The aim of this study was to characterize CD133 expression, and the distribution and morphological features of CD133(+) cells, in primary and secondary human central nervous system (CNS) neoplasms. METHODS AND RESULTS: Tumours were analysed by real-time reverse transcription polymerase chain reaction, western blot, flow cytometry and immunohistochemistry. Our results show that only small round blue cell tumours (SRBCTs) exhibit strong and consistent CD133 expression. Interestingly, glioblastomas, large-cell carcinomas and sarcomas were negative for CD133. Only glioblastomas with a focal small-cell component exhibited CD133 immunoreactivity in the SRBCT component. In addition, CD133 expression did not correlate with the expression of other markers associated with stem cell differentiation, including CD15 and nestin. CONCLUSIONS: CD133 expression in human CNS neoplasms is independent of the grade of malignancy but strongly correlates with SRBCT morphology. Together with recent findings showing that CD133 is quickly upregulated upon hypoxia and that CD133(-) cells can also exhibit stem cell properties, our data strongly question the suitability of CD133 as a brain tumour stem cell marker in vivo.