Schatz Stefanie, Willnow Lena, Winkels Monika, Rosengarten Jamila Franca, Theek Benjamin, Johnston Ian C D, Stitz Jörn
Research Group Medical Biotechnology and Bioengineering, Faculty of Applied Natural Sciences, University of Applied Sciences Cologne, Campusplatz 1, 51379 Leverkusen, Germany.
Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University Hannover, Calinstrasse 3-9, 30167 Hannover, Germany.
Antibodies (Basel). 2023 Sep 5;12(3):57. doi: 10.3390/antib12030057.
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.
用装饰有期望的膜锚定靶抗原（Ag）的高免疫原性病毒样颗粒（VLP）进行免疫接种时，可有效诱导产生靶特异性抗体。然而，例如，出于诊断目的，需要单克隆抗体（mAb）来对甲醛固定石蜡包埋（FFPE）活检组织样本进行组织学检查。为了产生FFPE抗原特异性单克隆抗体并作为概念验证（POC），我们首先建立了一种简化方案，仅对表达靶抗原神经生长因子受体（NGFR）的细胞进行甲醛和90°C热固定（FF90）。使用流式细胞术分析以及两种分别识别天然和FFPE抗原或仅识别天然抗原的单克隆抗体对FF90程序进行了验证。使用透射电子显微镜（TEM）和动态光散射（DLS）分析，源自HIV-1的展示天然和经FF90处理的C末端截短的NGFR（trNGFR）的VLP在颗粒形态上未显示出明显变化。随后用trNGFR装饰的FF90-VLP对小鼠进行重复免疫，并使用杂交瘤技术建立产生单克隆抗体的细胞克隆。在多轮筛选中，鉴定出九个细胞克隆，它们产生的单克隆抗体能特异性识别FF90和FFPE-NGFR中的表位。这种新方法的概念验证应有助于未来产生选择性靶向FFPE固定细胞表面抗原的单克隆抗体。
