Multiplex Cytokine Immunoassays — Minimizing Effort, Maximizing Results

It has been over 60 years since the first cytokine was identified. To date, more than 300 cytokines, chemokines, and growth factors have been studied with many functions involving the immune system (Turner et al. 2014). These cytokines often work in a network that modulates and regulates immune responses. Many of these cytokine molecules have an enigmatic role and in-depth research is essential to better understand the complexity of their association with various pathophysiological processes and the pleiotropic interactions that characterize the cytokine network.

Progress in mining these cytokine markers has shown that in drug discovery and the study of complex diseases, composite profiling of multiple biomarkers provides a more comprehensive understanding of drug effects and signatures in disease progression (Etzioni et al. 2003, Hsu et al. 2014, Williams 2009). The availability of commercial multiplex cytokine assays has greatly simplified this effort and enables end users to interrogate an entire network of cytokines in a single sample.

In small animal models, the ability to profile multiple cytokine markers using conventional single-plex ELISA techniques is often restricted by the requirement of a large sample volume. A typical 3-week-old mouse has a limited circulating blood volume of about 1.5–2.5 ml. Less than half of this volume is available in the form of plasma. This significantly limits the number of cytokine markers that can be studied per sample/animal. Consequently, studies that rely on only a few cytokines to determine whether they correlate with a physiological variable of interest may overlook the integrated effects of the complex cytokine network. In contrast, multiplex immunoassays offer a more comprehensive analysis whether for basic research, drug discovery, or monitoring of therapeutic interventions and provide considerable savings in both time and cost per assay.

While multiplex immunoassays continue to gain popularity in the fields of biomarker discovery and drug development, continuous improvement of these assays remains crucial in transitioning them to a mainstream role in research and eventually the clinical network. Here, we provide detailed insight into the SMARTS (Sensitivity, Multiplexibility, Actionable results, Reproducibility, Time, and Specificity) approach to better align user experience to the performance characteristics of these multiplex assays. The SMARTS concept has been implemented in product design, development, validation, and manufacturing of multiplex assays at Bio-Rad. The end goal of the SMARTS approach is to deliver end users with actionable results using the five performance characteristics of SMARTS to guide assay design and acceptance criteria. 

The most pressing driver for adopting an improved immunoassay platform is greater assay sensitivity and the ability to measure low abundance proteins from complex sample matrices. For the Bio-Plex Multiplex Immunoassay System, we design and develop multiplex assays with sub-picogram per milliliter limit of detection (LOD) and a wide dynamic range (4 log) that enables detection of healthy, disease, and stimulated samples in the same assay. A wide dynamic assay range significantly increases the utility of a multiplex assay as a protein detection and quantitation tool in diverse autoimmune, inflammatory, cancer, infectious, and malignant conditions. Since the development and validation of multiplex assays are very complex and time consuming, we’ve used our vast expertise to develop novel approaches to address multiplex cross-talk, optimize reagents to improve sensitivity, apply a clinical-like approach to improve reproducibility, and develop antibodies and antigens for greater specificity. In today’s fast-moving world, researchers demand tools to expedite sample analysis and conserve resources. The SMARTS approach ensures end users gain new insights into their research and spend less time on the bench.

Current status and future direction

Currently many commercially available multiplex cytokine immunoassays vary in their ability to measure serum and/or plasma cytokines (Alex et al. 2009, Berthoud et al. 2011, Breen et al. 2011, Christiansson et al. 2014). Often, the evaluation and comparison between different commercial assays are limited by the lack of a reference standard. Despite these known hurdles, the measurement of multiple cytokines simultaneously within a specimen still enables the assessment of relative levels of cytokines within a system. Multiplex assays have become widely used in basic research because of their many advantages over single-plex ELISA assays. The benefits include high throughput, lower cost, and low consumption of sample volume. These assays are reaching a stage of maturity where, with proper validation, they can be successfully implemented in clinical development programs. While the utility of multiplex cytokine assays as diagnostic and prognostic tools in diverse autoimmune, inflammatory, infectious, and malignant conditions is still limited, its potential is expanding. Relative to conventional ELISA assays, many of these assays offer a wider assay range and have become increasingly more sensitive (down to the single digit picogram/ml range). Aside from the Luminex xMAP platform, the most desired immunoassay module researchers want access to is an open platform with enhanced speed and an automated solution for generic immunoassay applications that can be adapted for future requirement changes. At present, suspension immunoassay is the prevailing technology for FDA-cleared multiplex protein measurements (Tighe et al. 2015).

Learn more about Bio-Plex Multiplex Immunoassay technology.

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