Protocol

Clinical Applications of Capillary Electrophoresis

Volume 919 of the series Methods in Molecular Biology pp 233-248

Date:

Chip-Based Immunoassays

  • Akwasi A. AporiAffiliated withDepartment of Bioengineering, University of California Berkeley
  • , Amy E. HerrAffiliated withDepartment of Bioengineering, University of California Berkeley Email author 

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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.

Key words

Microfluidic immunoassay Immunosubtraction Sample preparation PAGE Microfabrication