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: takayama@gatech.edu

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.