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Related Concept Videos

Ligand Binding Sites02:40

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Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
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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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Related Experiment Video

Updated: May 2, 2026

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
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Ligand screening using enzymatic assays.

Kiira Ratia1, Shahila Mehboob, Hyun Lee

  • 1Research Resources Center and the Departments of Medicinal Chemistry and Pharmacology, University of Illinois at Chicago, Chicago, IL, USA, kratia@uic.edu.

Methods in Molecular Biology (Clifton, N.J.)
|March 5, 2014
PubMed
Summary

Enzyme assays enable high-throughput screening of small molecules to identify potential drug candidates. This process involves optimizing assay conditions, executing the screen, and analyzing data to validate enzyme inhibitors, exemplified by Hepatitis C virus NS3/4A protease.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Drug Discovery

Background:

  • Enzymatic assays are crucial for quantifying ligand effects on enzyme activity.
  • Enzyme-based assays are well-suited for high-throughput screening (HTS) of small molecules.
  • Optimization of assay kinetics, components, signal, and stability is essential for HTS.

Purpose of the Study:

  • To detail the stages of enzyme-based ligand screening.
  • To illustrate the HTS process using Hepatitis C virus NS3/4A protease as a model target.
  • To emphasize the importance of optimization and validation in HTS.

Main Methods:

  • Optimization of enzyme assay kinetics, components, readout signal, and stability.
  • Consideration of replicates, plate layout, compound concentration, and assay assembly for screening.
  • Data processing, analysis, and secondary/tertiary assays for hit validation.

Main Results:

  • Enzyme assays provide quantifiable data on ligand-enzyme interactions.
  • HTS allows for rapid profiling of numerous small molecules against a target.
  • A systematic approach is required for successful ligand screening and hit identification.

Conclusions:

  • Enzyme assays are a powerful tool for drug discovery and HTS.
  • Rigorous optimization and validation are critical for reliable screening results.
  • The described methodology is applicable to various enzyme targets, including viral proteases.