Development of Microscale Immunoassays using Aqueous Two-Phase Systems

Abstract

Aqueous two-phase systems (ATPSs) have been traditionally applied in liquid-liquid extraction processes to purify and recover biological products. Due to their high biocompatibility, easy handling, and potential to be scaled up, ATPSs have also been applied in nonconventional innovative applications, such as solution microarrays to improve performance and cost-effectiveness of immunoassays. The enzyme linked immunoassay (ELISA) has numerous applications in clinics, industrial R&D, environmental surveillance, forensics, and academic research. However, the high cost of biologic reagents, such as antibodies, and relatively time-consuming protocols represent significant drawbacks. The enzyme-linked immunospot (ELISpot) is a cell-based assay widely used to evaluate vaccine formulations in vitro. However, it requires considerable amounts of expensive vaccine reagents. In this thesis, two innovative ATPS-based platforms were developed to reduce the amount of capture and detection antibodies required for single sandwich ELISA and to minimize the amount of vaccine reagents required for vaccine formulation screening by ELISpot. An ATPS composed of poly (ethylene glycol) (PEG) and dextran was used to confine antibodies for multiplex analyte detection in the so-called ATPS-ELISA, which consumed 3-fold less capture and 2.5-fold detection antibodies compared to conventional sandwich ELISA. The novel PEG-bovine serum albumin (BSA) system was used to confine immune cells and vaccine reagents in microdroplet reactors, which enabled screening hemagglutinin (antigen) and Toll-like receptors (TLR) agonists (adjuvants), using only a fifth of the amounts required by conventional ELISpot. Therefore, the ATPS-based techniques presented in this thesis can improve the cost-effectiveness of immunoassays such as ELISA and ELISpot. In both cases, the consumption of expensive reagents has been significantly reduced, while producing comparable performance characteristics compared to conventional assays. Thus, ATPS-based methods have high potential for the development of innovative immunoassay technologies.