Multiplex bead binding assays using off-the-shelf components and common flow cytometers

J Immunol Methods. 2021 Mar;490:112952. doi: 10.1016/j.jim.2020.112952. Epub 2020 Dec 25.


The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.

Keywords: Affinity determination; Antibody screening; Antibody-antigen interaction; Multiplex assay; Phage display.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / metabolism
  • Biotin / analogs & derivatives
  • Biotin / metabolism
  • COVID-19 / diagnosis*
  • COVID-19 Serological Testing / methods*
  • Cell Surface Display Techniques
  • Flow Cytometry
  • Fluorescent Dyes
  • HEK293 Cells
  • High-Throughput Screening Assays
  • Humans
  • Immunomagnetic Separation
  • Microspheres
  • Mutation / genetics
  • Protein Binding
  • SARS-CoV-2 / genetics
  • SARS-CoV-2 / metabolism*
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / metabolism*


  • Bacterial Proteins
  • Fluorescent Dyes
  • Spike Glycoprotein, Coronavirus
  • biotin-streptavidin complex
  • spike protein, SARS-CoV-2
  • Biotin