One-incubation one-hour multiplex ELISA enabled by aqueous two-phase systems

Analyst. 2020 May 18;145(10):3517-3527. doi: 10.1039/d0an00383b.


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 image processing software as an ImageJ plugin is also described and provided.

MeSH terms

  • Cell Line
  • Dextrans / chemistry
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Horseradish Peroxidase / metabolism
  • Humans
  • Polyethylene Glycols / chemistry
  • Time Factors
  • Water / chemistry*


  • Dextrans
  • Water
  • Polyethylene Glycols
  • Horseradish Peroxidase