Abstract
Microfluidic immunoassay techniques offer advantages in speed, automation, and portability over bench-top gold standard counterparts. In particular, on-chip immunosubtraction is a rapid homogeneous immunoassay used for reporting both protein native mobility and binding specificity. Immunosubtraction is performed by removing antibody-bound target proteins from electrophoretic detection via a size-based exclusion filter, while unbound nontarget proteins are able to pass through the filter for downstream detection. Immunosubtraction is achieved on-chip by fabrication of discrete patterned polyacrylamide (PA) gel regions. Additionally, PA gel regions are used to define on-chip sample preparation regions for protein enrichment, fluorescent labeling, and antibody-target binding prior to immunosubtraction. Here we describe the immunosubtraction device fabrication technique as well as the electrophoretic assay protocol for determining target protein mobility and binding specificity within complex biological samples including cerebrospinal fluid.
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Acknowledgments
This material is based upon the work supported by the Defense Advanced Research Projects Agency (DARPA) under Award N66001-09-1-2118 (to A.E.H.) and the National Institutes of Health through the NIH Director’s New Innovator Award Program (1DP2OD007294 to A.E.H.). A.A.A. is a National Defense Science and Engineering Graduate (NDSEG) Fellow and UNCF/Merck Fellow. A.E.H. is an Alfred P. Sloan Foundation Research Fellow in chemistry.
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Apori, A.A., Herr, A.E. (2013). Chip-Based Immunoassays. In: Phillips, T., Kalish, H. (eds) Clinical Applications of Capillary Electrophoresis. Methods in Molecular Biology, vol 919. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-029-8_21
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DOI: https://doi.org/10.1007/978-1-62703-029-8_21
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