ANYL 321 |
| Major advances in the disease diagnostics are likely to come from the approach of systems biology. Systems biology is to use high-throughput methods to take a global approach to study the interconnections of the many biochemical pathways carried out by proteins or metabolites in cells that work together in an organism. High-throughput, affinity-based methods with fluorescence or chemiluminence detection offer sensitivity as low as femtomolar, and have become indispensable tools for proteomics and systems biology. The affinity-based immunoassays, however, critically depend on the quality of the affinity reagents, such as antibodies, peptides, DNA/RNA aptamers. In this study, recombinant interleukin-4 (IL-4) was chosen as a model protein analyte. Three kinds of monoclonal antibodies with different epitope and specificity against the same IL-4 were used to quantify IL-4 using ‘sandwich-type' (antibody-antigen-antibody) immunoassays and three different proteomic platforms, protein microarrays, suspension arrays, and surface plasmon resonance (SPR). More precisely, the immobilized capture antibody was used to capture the analyte. The bound analyte was subsequently recognized by biotinylated antibody with a different epitope that was identified by streptavidin-phycoerythrin conjugate. Since SPR is a direct label-free measure of antibody-antigen mass and does not require a fluorogenic substrate to produce a signal, antibody with a different epitope regarding the capture antibody was used as the detection antibody. We examined the sensitivity, dynamic range, and reproducibility associated with each platform. Normal mouse serum with spike-in recombinant IL-4 was also used to examine the robustness of the measurement platforms. |
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Biomarker Discovery
1:30 PM-4:20 PM, Monday, August 20, 2007 BCEC -- 104A, Oral
Division of Analytical Chemistry |