Abstract C21: Evaluation of an 8-plex cytokine immunoassay on the AVANTRA®, a novel automated biomarker workstation

Abstract

Abstract Decision Biomarkers, Inc. (DBI) has developed a novel, automated, immunoassay platform to simultaneously evaluate multiple biomarkers in a single reaction vessel. Biomarker panels are used for many research and clinical indications. We have generated data from a multiplex assay measuring a panel of 8 cytokines: IL-1β, IL-2, IL-5, IL-6, Il-8, IL-10, IL-12, and TNFα. The heart of the system is a microarray of the biomarker specific monoclonal antibodies spotted onto a glass slide which has been coated with an ultra-thin, two-dimensional film of nitrocellulose. This microarray is assembled in a plastic biochip (MAX BIOCHIP™): a self-contained device with a series of micro fluidic channels, valves, sensors, and reagent chambers containing dried, biotinylated detection antibodies, streptavidin-conjugated Cy3, and wash buffer. The biochip is processed in the AVANTRA® biomarker workstation following injection of 200 µl of sample (100 µl of plasma diluted in 100 µl of PBS). The instrument initiates, in sequence, the flow of sample, detection antibody, label, and buffer, across the array. The end result is in a fluorescent labeled complex that is then imaged by an internal CCD camera. Spot intensity is correlated with analyte concentration by instrument software. “Master” curves” electronically stored in the instrument are used to calculate analyte concentrations through a 2-point calibration scheme. Each cytokine assay has a 4-log dynamic range and individual analytical sensitivities ranging between 2.7 and 20 pg/ml. Both the intra- and interassay precision results yielded mean CV's consistently <10% Reproducibility was demonstrated by overlapping curves following replicate standard evaluation. The unique surface chemistry, marker specific reagents, and enhanced biochip micro fluidics and instrument automation, all combine to produce a robust and easy to use multiplex immunoassay. Currently in development are a 10-plex angiogenesis panel and a 12-plex cardiac panel that will soon be available. Additionally, assays for numerous other biomarker panels are slated for future development. Custom assay development is also available. In summary, DBI has developed a sensitive and precise, multiplex immunoassay for 8 cytokines that can run on its new, automated platform, the AVANTRA® biomarker workstation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C20.