Mintra Tongdee a,b, Cameron Yamanishi b, Midori Maeda b, Taisuke Kojima b, John Dishinger c, Rattikan Chantiwas a and Shuichi Takayama *,b

a Department of Chemistry and Center of Excellence for Innovation in Chemistry and Flow
Innovation-Research for Science and Technology Laboratories (FIRST Labs), Faculty of Science,
Mahidol University, Rama VI Rd., Bangkok 10400, Thailand
b Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta 30332,
Georgia, USA
c PHASIQ, Inc., Ann Arbor, Michigan 48109, USA
*Corresponding author:

This work describes a convenient one-hour enzyme-linked immunosorbent assay (ELISA) formulated with conventional antibodies and horseradish peroxidase (HRP) reagents. The method utilizes aqueous two-phase system (ATPS) droplet formation based on poly(ethylene glycol) (PEG)-containing sample solution triggered rehydration of dehydrated dextran (DEX) spots that contain all antibody reagents. Key advances in this paper include development of a formulation that allows a quick 1-hour overall incubation time and a procedure where inclusion of the HRP reagent in the PEG solution reduces the number of washing and incubation steps required to perform this assay. As an assay application, a 5-plex cytokine test compares cytokine secretion of differentially-treated human ThP-1 macrophages. Given the use of only readily available reagents and a common Western blot imaging system for the readout, this method is envisioned to be broadly applicable to a variety of multiplex immunoassays. To facilitate broader use, companion imageprocessing software as an ImageJ plugin is also described and provided.

M. Tongdee, C. Yamanishi, M. Maeda, T. Kojima, J. Dishinger, R. Chantiwas, S. Takayama. Oneincubation one-hour multiplex ELISA enabled by aqueous two-phase systems. Analyst (2020), 145,
3517-3527. DOI: 10.1039/d0an00383b