A mix-and-read cell-based assay for antibody screening against Epithelial Growth Factor Receptor
Poster May 08, 2013
Wayne P Bowen, David Onley, Paul Wylie, Diana Caracino and Tristan Cope
Antibodies against a wide range of protein targets have been either approved or are currently under development for therapeutics. The use of flow cytometry to identify antibody-antigen binding has been well characterised. While flow cytometry has been widely utilised, it requires the use of suspension cells or adherent cells that are removed from the well, so it is unable to analyse cells in situ. In addition, as the cells are passed through a flow tube, low affinity antibodies can dissociate from the antigen as the equilibrium of antibody concentration to antigen is changed. Flow cytometry is typically not a high throughput method for this kind of assay. Though higher-throughput systems are now currently available, they still have to remove cells from a plate which can lead to cross-contamination, and require wash steps which are not ideal for low-affinity antibodies. We offer an alternative high-throughput method for looking at antibody binding whereby the primary screen can be run through the mirrorball® high sensitivity cytometer to rapidly identify antibodies of interest.
Here we present a sensitive robust, mix-and-read method for the screening of antibodies against cell surface proteins. The mirrorball® high sensitivity cytometer is used to quantify the cellular fluorescence in cultures in 384-well microplates. We describe its use for the determination of human epithelial growth factor receptor (EGFR) antibody binding in A549 cells which are known to express high levels of EGFR. A549 cells were incubated with mouse anti-EGFR antibody and fluorescently-labelled anti-mouse IgG antibody. Without washing away unbound antibodies, plates were scanned and fluorescence of each cell quantified. Clear concentration-dependent antibody binding was observed with low assay variability. Addition of Vybrant™ DiO, a 488 nm-excitable cell stain, allowed detection of cells irrespective of the amount of anti-EGFR antibody binding for improved assay performance. With its simple operation, no-wash format, and high sensitivity, this new method is well-suited for high throughput antibody screening.
Cerebral Malaria Insights: Pathogenesis, Host Parasite Interactions including Host ResistancePoster
Cerebral malaria is a dreadful disease transmitted by mosquito. The major preventive approach is focused more in vector control than development of anti-malarial drug. The purpose of this presentation is to analyze different aspects of disease manifestations including clinical symptoms and pathogenesis in the context of mosquito borne infections in different geographical regions of the world.READ MORE
Amoebic Meningoencephalitis: Etiology, Infection and PreventionPoster
The presentation covers the different types of organisms that can cause amoebic meningioencephalitis. The two main types focused on are primary amoebic meningioencephalitis and granulomatous amoebic meningioencephalitis. The method of transmission and target hosts are vastly different. Prevention of these diseases is imperfect at best, as complete avoidance is the only way to not contract the disease.READ MORE
Depressive Symptoms Related to Biological Markers of Immune Functioning among Young People with HIVPoster
Youth living with HIV (YLWH) are at risk for depression. This study aimed to identify trends in depressive symptoms for YLWH in a specialty-care clinic and follow up clinical treatment procedures.READ MORE