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Abstract
Decision Biomarkers has developed technology to coat a standard glass slide with a thin film (<1 micron) of nitrocellulose. This thin film provides the benefits of nitrocellulose in protein assays, but offers a unique, low-background, high-affinity environment for performing fluorescent-based protein assays. We will demonstrate the performance of these slides in an immunoassay format, and compare the results to commercially available substrates. We show a simple, easy to use surface requiring no protein modification and no preprocessing steps that is useful in detecting low abundant proteins due to its inherently low fluorescent background.
Abstract
Decision Biomarkers, Inc. (DBI) has developed an automated immunoassay platform to simultaneously evaluate multiple biomarkers in a single test sample. As initial proof of principle, we have generated data from a multiplex assay measuring a panel of 3 cytokines: IL-1bß, IL-6, and TNF-α. In brief, specific monoclonal antibodies (MAbs) are individually arrayed in a rectangular pattern on a glass slide coated with an ultra-thin, two-dimensional film of nitrocellulose (NC). The slide is then assembled in a plastic biochip: a self-contained device with a series of microfluidic channels, valves, sensors, and reagent chambers containing biotinylated detection polyclonal antibodies (pAbs), streptavidin-conjugated Cy3, and wash buffer. The biochips are placed in the AVANTRA™Q400 biomarker workstation instrument and samples are injected. The instrument initiates, in sequence, the flow of serum, detection antibody, fluorescent label, and buffer, across the array, resulting in a fluorescently labeled complex. An internal CCD camera then images the labeled array and fluorescent output is correlated with analyte concentration. Each individual cytokine assay had a dynamic range up to 5000 pg/ml, with an analytical sensitivity of < 10 pg/ml. The inter-assay precision yielded mean CV’s consistently under 10%. Reproducibility was evaluated by replicate evaluation of the standards, resulting in overlapping working curves. In summary, DBI has developed a sensitive and precise, multiplex immunoassay for 3 cytokines that can run on its platform, the AVANTRA™Q400 biomarker workstation.
Abstract
Decision Biomarkers, Inc. 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-1b, IL-2, IL-6, Il-8, IL-10, IL-12, IFNg and TNFa. 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 microfluidic channels, valves, sensors, and reagent chambers containing dried, biotinylated detection antibodies, streptavidin-conjugated Cy3, and wash buffer. The biochip is processed in the AVANTRA™Q400 biomarker workstation following injection of 200 ml of sample (100 ml of plasma diluted in 100 ml 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 fluorescently labeled complex that is then imaged by an internal CCD camera
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