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Related Concept Videos

Enzyme-Linked Immunosorbent Assay01:33

Enzyme-Linked Immunosorbent Assay

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In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane.
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen.  Enzyme-substrate reaction allows the antigen to be visualized or...
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One-incubation one-hour multiplex ELISA enabled by aqueous two-phase systems.

Mintra Tongdee1, Cameron Yamanishi2, Midori Maeda2

  • 1Department 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 and Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta 30332, Georgia, USA.

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Summary

A new one-hour enzyme-linked immunosorbent assay (ELISA) uses aqueous two-phase systems (ATPS) for rapid, multiplexed cytokine detection. This simplified method offers broad applicability for various immunoassays.

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Area of Science:

  • Biochemistry
  • Immunology
  • Assay Development

Background:

  • Traditional enzyme-linked immunosorbent assays (ELISA) often require lengthy incubation and multiple washing steps.
  • Multiplexed immunoassays are valuable for simultaneous detection of multiple analytes, but can be complex to develop and perform.
  • There is a need for faster, more streamlined immunoassay formats suitable for diverse applications.

Purpose of the Study:

  • To develop a rapid, one-hour enzyme-linked immunosorbent assay (ELISA) using aqueous two-phase system (ATPS) droplet formation.
  • To simplify the assay procedure by reducing incubation and washing steps.
  • To demonstrate the assay's utility for multiplexed cytokine detection and provide accompanying image analysis software.

Main Methods:

  • Formulation of dehydrated dextran (DEX) spots containing antibody reagents, rehydrated by poly(ethylene glycol) (PEG)-containing sample solutions.
  • Integration of horseradish peroxidase (HRP) reagent into the PEG solution to minimize assay steps.
  • Development of an ImageJ plugin for image processing and data analysis of multiplexed assays.

Main Results:

  • Achieved a complete assay time of one hour, significantly reducing traditional ELISA protocols.
  • Demonstrated a 5-plex cytokine test on differentially-treated human ThP-1 macrophages, showcasing multiplexing capability.
  • Validated the use of readily available reagents and a standard Western blot imaging system for readout.

Conclusions:

  • The developed ATPS-based ELISA offers a convenient and rapid method for multiplexed immunoassays.
  • The simplified protocol and use of common equipment make this assay broadly applicable and accessible.
  • The provision of image processing software further facilitates the adoption and use of this novel assay format.