Multiplexed particle-based flow cytometric assays

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Abstract

Several methods have been developed to quantify soluble analytes in biological fluids and tissue culture samples, including bioassays, ELISA, RPA and PCR. However, each of these techniques possesses one or more significant limitations; ELISA will only measure one analyte as a time; PCR does not detect native protein. The recent development of particle-based flow cytometric assays has raised hopes that many of these limitations can be overcome. The technology utilizes microspheres as the solid support for a conventional immunoassay, affinity assay or DNA hybridization assay which are subsequently analyzed on a flow cytometer. Several multiplexed bead systems are currently marketed by different vendors. We have used the Luminex FlowMetrix™ system which consists of 64 different bead sets manufactured with uniform, distinct proportions of red and orange fluorescent dyes (detected by FL2/FL3 on a FACScan). Each bead set forms the basis of an individual assay using a green fluorescent reporter dye (FL1). This system facilitates the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. They can also be developed into rapid, ‘no wash’ assays that can be completed in <2 h. This review traces the historical association between microspheres and flow cytometry, the development and use of particle-based flow cytometric assays, how they compare with current assays and potential future developments of this very exciting technology.

Section snippets

The desire to quantify

Analytical assays are the cornerstone of biomedical research and medical diagnosis. A bewildering array of methodologies has been employed to quantify soluble analytes, and significant improvements have been made in assay performance in the last 20 years. Despite the emergence of DNA-based assays, methodologies that quantitate the native analyte still form an important part of medical diagnostics. However, it is clear that many improvements can still be made. As our desire to quantitate

Microspheres and flow cytometry

In this review, I will focus on the emergence of an exciting new methodology that I believe will become a primary assay platform in the future: particle-based flow cytometric assays. To my knowledge, only three companies offer this technology; Luminex (www.luminexcorp.com), Becton-Dickinson (www.bdfacs.com or www.pharmingen.com; due for release spring/summer 2000) and DiaSorin (www.diasorin.com). While each system will be discussed in this review, we will focus on the Luminex technology as that

In search of the perfect assay

In order to discuss recent developments in particle-based flow cytometric assays, we first need to provide a framework for determining the advantages and disadvantages of these assay systems with currently available methods. This will also prove useful in discussing improvements that could be made in the future. So how do you develop the ‘perfect’ assay? Here are six key considerations:

  • 1.

    Specificity: many protein-based assays rely on the exquisite specificity of antibodies and it seems likely

Cytokines: a test bed for assay development

While a variety of analytes will be discussed in this review, cytokines have been the focus of our own attempts to develop a better assay system. While they will feature prominently here, I believe the lessons learnt will be applicable to the quantitation of many other analytes. Cytokines play a central role in the regulation of hematopoiesis; mediating the differentiation, migration, activation and proliferation of phenotypically diverse cells (Mosmann and Coffman, 1989; O’Garra and Murphy,

Development of a multiplexed cytokine assay

We have used the FlowMetrix™ System (Luminex, Austin, TX) to develop an assay that could quantitate multiple cytokines simultaneously at the protein level (Carson and Vignali, 1999). Although much of this review focuses on this technology, many of the comments detailed below are likely to apply to any future particle-based flow cytometric assay developed by other vendors. This system uses microspheres as the solid support for a conventional immunosorbent assay with a green-fluorescent reporter

Use of multiplexed particle-based flow cytometric cytokine assays

We have used our FlowMetrix™ assay to quantify cytokines in a variety of systems. We have previously shown that disruption of the transcription factor STAT4 blocks TH1 development, while deletion of STAT6 blocks TH2 development (Shimoda et al., 1996; Thierfelder et al., 1996). We have used this assay to further analyze cytokine production by TH1 and TH2 cells from STAT 4 and/or STAT6 deficient mice (Fig. 2). Splenic CD4+ T cells were stimulated with anti-CD3 and anti-CD28 under conditions

Variations on a theme

A number of other investigators have used the FlowMetrix™ technology or similar approaches to develop multiplexed assays for a diverse array of analytes. Kellar and colleagues (personal communication) have adapted the Luminex bead technology for the detection of human immunoglobulins, anti-viral antibodies and viral antigens (Fig. 3B,E,F). In collaboration with Abbas Vafai and Scott Schmid, Kellar’s group have developed an assay to detect anti-varicella zoster virus (VZV) human IgG and VSV

The future

I believe future research and technology development is likely to focus on four main areas.

  • 1.

    Technology development: it is likely that significant improvements will be made in bead chemistry, instrumentation and software. It is also likely that other companies will develop their own versions of this technology, particularly those that are already in the flow cytometry market.

  • 2.

    Improved antibodies: antibodies possess the remarkable qualities of recognizing native proteins with high specificity and

Acknowledgements

I wish to dedicate this review to the memory of Richard Carson, who developed the FlowMetrix™ cytokine assay in my lab. I am indebted to the many investigators who kindly provided data (much of it unpublished) for this review: Marcia Blackman, Kathi Kellar, Francis Mandy, Scott Schmid, Erkki Soini, Ralph Tripp, Abbas Vafai and Vance Vorndam. Our work was supported by the NIH (AI-39480), a Cancer Center Support CORE grant (5 P30 CA-21765) and the American Lebanese Syrian Associated Charities

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