Cytometric bead array: a multiplexed assay platform with applications in various areas of biology

doi:10.1016/j.clim.2003.11.017

Clinical Immunology

Volume 110, Issue 3, March 2004, Pages 252-266

https://doi.org/10.1016/j.clim.2003.11.017 Get rights and content

Abstract

The introduction of flow cytometric bead-based technology has added a new approach for investigators to simultaneously measure multiple analytes in biological and environmental samples. This new technology allows for (1) evaluation of multiple analytes in a single sample; (2) utilization of minimal sample volumes to glean data; (3) reproducibility and results comparative with previous experiments; (4) direct comparison with existing assays; and (5) a more rapid evaluation of multiple samples in a single platform. The cytometric bead array (CBA) system enables simultaneous measurement of multiple analytes in sample volumes too small for traditional immunoassays. Results have been presented for the analysis of a variety of human cytokines. In addition, the technology allows for the design and creation of assays to measure a variety of analytes including inflammatory mediators, chemokines, immunoglobulin isotypes, intracellular signaling molecules, apoptotic mediators, adhesion molecules, and antibodies. New initiatives put forward by the Human Genome Project and the FDA require the development and use of assays for the rapid simultaneous quantitation of multiple analytes. The CBA technology provides the ability to quantify multiple proteins within a given sample, with precision and consistency.

Section snippets

Introduction to cytometric bead-based assays

The ability to measure soluble mediators involved in immune regulation has been the focus of researchers for over three decades. To this point, immunologists and cell biologists have focused on the in vivo and in vitro measurement of multiple analytes and their associated receptors. The importance of assessing the relative levels of these mediators is evidenced by the fact that they form the basis of a sophisticated cellular communication network for normal immune function as well as in disease

Measurement of cytokines

The direct quantitation of cytokines and chemokines from samples has been problematic. We have developed multiple flow cytometric bead-based immunoassays that are capable of measuring the levels of up to seven different analytes simultaneously, from a single serum, plasma, or cell culture supernatant sample. Particles (7.5 μm in diameter) are dyed with different fluorescence intensities. The dye, incorporated in the beads fluoresces strongly at 650 nm (measured as FL3 signals in BD FACScan and

Measurement of complement-derived inflammatory mediators

The complement (C) system is a major component of innate immune function. The involvement of complement in the pathogenesis of antigen–antibody-based autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and acute glomerulonephritis is well documented [22], [23], [24], [25]. Other pathologies include sepsis [26], [27], adult respiratory distress syndrome (ARDS) [28], and multiorgan failure [29]. More recently, activation of the C cascade has been implicated

Measurement of intracellular signaling molecules

Stimulation of cells through receptors leads to the activation of intracellular signaling pathways, which in turn leads to many different cellular responses. A common feature of signaling pathways, especially in the immune system, is the activation of kinases, which phosphorylate either tyrosine or serine/threonine residues found on a series of other proteins. Many kinases themselves are activated by being phosphorylated. This leads to a signaling cascade or amplification of the response.

Measurement of apoptosis

Apoptosis, or programmed cell death, is necessary for tissue homeostasis, and its perturbation can lead to multiple disease states [51]. Signaling via apoptosis can generally be divided into receptor- and mitochondrial-mediated pathways. These pathways converge at several downstream points including the mitochrondia, caspase activation, and substrate cleavage [52]. When apoptosis is induced, procaspases are proteolytically cleaved and reassembled to form active heterotetrameric enzymes. The

Detection and quantitation of specific antibodies

The bead-based assay system can be easily adapted to carry out immunoassays for detecting antibodies to various antigens either in serum or culture supernatants. This can be accomplished by following variations of assay design as described for other analytes. In one approach, antigens that are amenable to immobilization on solid surfaces could be conjugated covalently or in a noncovalent manner to the beads. Incubation of antigen-coated beads with any biological sample of interest, serum, CSF,

Multiplex bead-based immunoassay data analysis

Bead-based immunoassays such as the CBA are powerful tools to measure multiple analytes (e.g., proteins and mRNAs) within individual samples. Multiparameter analysis of the large amounts of data generated by bead-based assays provides challenges to the investigator. This data must be quickly and efficiently reanalyzed and converted from a general list of characteristics about the sample to specific, useful, and comparable results for individual analytes. Without a rapid and simple system to

Acknowledgements

The authors wish to acknowledge the following for their contribution: Brandon Bowman, Michael Boyer, Ada Chan, Kifle Tzeggai, William K. Collins, Jian Gu, John King, Hai Le, Feng-Jun Luan, Teresa Giago-McGahan, Anne Parsons, Frank Vegh.

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Cytometric bead array: a multiplexed assay platform with applications in various areas of biology

Abstract

Section snippets

Introduction to cytometric bead-based assays

Measurement of cytokines

Measurement of complement-derived inflammatory mediators

Measurement of intracellular signaling molecules

Measurement of apoptosis

Detection and quantitation of specific antibodies

Multiplex bead-based immunoassay data analysis

Acknowledgements

J. Immunol. Methods

Methods Cell. Biol.

J. Immunol. Methods

Hum. Immunol.

Mol. Cell. Probes

Mol. Cell. Probes

Genomics

J. Immunol. Methods

Am. J. Med.

Immunopharmacology

Immunol. Lett.

Lancet

J. Allergy Clin. Immunol.

Trends Immunol.

Gene

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Homogeneous immunofluorometric assays of alpha-fetoprotein with macroporous, monosized particles and flow cytometry

Clin. Chem.

Immunoreactive bead (IRB) assay for the quantitative and simultaneous flow cytometric detection of multiple soluble analytes

Cytometry

T-lymphocyte functionality assessed by analysis of cytokine receptor expression, intracellular cytokine expression, and femtomolar detection of cytokine secretion by quantitative flow cytometry

Cytometry

A new flow cytometry-based multi-assay system: 1. Application to cytokine immunoassays

Cytometry

Simultaneous quantification of six human cytokines in a single sample using microparticle-based flow cytometric technology

Clin. Chem.

Method and apparatus for distinguishing multiple subpopulations of cells, United States Patent 4717655

The sensitive detection and quantitation of antibody to HCV by using a microsphere-based immunoassay and flow cytometry

Cytometry