资源 - 科学海报
Automated High-Throughput Functional Cell-based Bioassays for Blocking Antibodies下载
May 04, 2012
Authors: Cameron Pride, Sunitha Sastry, DiscoveRx Corporation, Fremont, California; Brad Larson,James Davis, and Peter Banks, BioTek Instruments, Inc., Winooski, Vermont
Receptor tyrosine kinases (RTK) are cell-surface receptors that are critical in normal cell signaling processes and are implicated in a variety of disease areas such as cancer, inflammation, diabetes, CNS and others. Mutations leading to overexpression of Epidermal Growth Factor Receptor (EGFR), in particular, have been linked to colon and lung cancers, among others. Monoclonal antibodies against receptor tyrosine kinases have revolutionized cancer therapeutics due to their highly selective therapeutic effect while having minimal off-target effects (Imai and Takaoka 2006). Many FDA-approved monoclonal antibodies, whose mechanisms of action target kinases, exist as therapeutic agents. However, various low throughput strategies such as ELISA or complex in-vivo models are often utilized to identify anti-ligand, and anti-receptor antibodies. Therefore, functional cell-based assays that can detect antagonistic activity of blocking antibodies and instrument platforms that can automate and demonstrate sensitive detection are highly desirable in therapeutic antibody discovery and characterization.
In this study we describe a novel, target-specific cell-based assay platform that can be used to detect and differentiate potential inhibitors of Epidermal Growth Factor receptors. The assay uses a full-length receptor protein allowing for detection of antibody inhibitors for ligands of membrane bound receptors and is based on a proprietary Enzyme Fragment Complementation (EFC) Assay Technology denoted as PathHunter® Receptor Tyrosine Kinase Functional assay. The PathHunter assay enables antibody interrogation and characterization in a cellular context. The assay was automated in 384-well format using simple, modular contact and non-contact instrumentation for fast and efficient creation of dose-response curves, as well as cell and reagent dispensing. Detection of the chemiluminescent signal was carried out using a multi-mode microplate reader with integrated data analysis. Optimization, validation, and pharmacology data demonstrate how the combination of assay technology and instrumentation create an ideal method for the detection of blocking antibodies in a convenient, reproducible manner that can save valuable time and translates routine bench top assays to high throughput methodologies.