Multiplexed microbead immunoassays by flow cytometry for molecular profiling: Basic concepts and proteomics applications

Abstract

Flow cytometry was originally established as an automated method for measuring optical or fluorescence characteristics of cells or particles in suspension. With the enormous increase in development of reliable electronics, lasers, micro-fluidics, as well as many advances in immunology and other fields, flow cytometers have become user-friendlier, less-expensive instruments with an increasing importance for both basic research and clinical applications. Conventional uses of flow cytometry include immunophenotyping of blood cells and the analysis of the cell cycle. Importantly, methods for labeling microbeads with unique combinations of fluorescent spectral signatures have made multiplex analysis of soluble analytes (i.e. the ability to detect multiple targets in a single test sample) feasible by flow cytometry. The result is a rapid, high-throughput, sensitive, and reproducible detection technology for a wide range of biomedical applications requiring detection of proteins (in cells and biofluids) and nucleic acids. Thus, novel methods of flow cytometry are becoming important for diagnostic purposes (e.g. identifying multiple clinical biomarkers for a wide range of diseases) as well as for developing novel therapies (e.g. elucidating drug mechanisms and potential toxicities). In addition, flow cytometry for multiplex analysis, coupled with automated sample handling devices, has the potential to significantly enhance proteomics research, particularly analysis of post-translational modifications of proteins, on a large scale. Inherently, flow cytometry methods are strongly rooted in the laws of the physics of optics, fluidics, and electromagnetism. This review article describes principles and early sources of flow cytometry, provides an introduction to the multiplex microbead technology, and discusses its applications and advantages in comparison to other methods. Anticipated future directions, particularly for translational research in medicine, are also discussed.

Keywords::

• Flow cytometry
• immunoassay
• multiplex
• biomarker
• proteomics

Acknowledgements

The authors acknowledge the many helpful discussions during the preparation of this article with both Drs. Kim Janatpour and Ralph Green. V.V.K thanks Dr. Venkat Venkateswaran for his expert knowledge in many aspects of the multiplex assay development and for many valuable discussions on this topic. He is also acknowledged for providing the data used in Figure 4. Multiplex biomarker research for P.A.L. and I.H.K. is supported in part by NIH grants (Pacific Southwest Center for Biodefense and Emerging Infectious Diseases U54-AI065359, R24-RR022907, and the Base Grant to the California National Primate Research Center RR00169), a USAID grant (PGA-7251-07-001), and by funds from the Department of Pathology and Laboratory Medicine at U.C. Davis. P.A.L. is principal investigator of a research contract between U.C. Davis and the Millipore Corporation (Billerica, MA). V.V.K. is supported by an NIH grant: Research Infrastructure for Minority Institutions P20MD002732.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.