Saving Time and Sample - Drug Discovery and Development

Technology In Action

Researchers step up to multiplex bead-based platforms to detect proteins for target indentification through clinical analysis.

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Figure 1: Measurement of biomarkers is crucial to virtually every step of the drug discovery and development process. (Source: David Hayes, Millipore)

In the post-genomic era of drug discovery and development, success hinges upon characterizing relevant proteins functioning in multiple biological pathways and complexes. Drug discovery research is being pushed towards increasing scale and complexity. Measuring proteins one at a time using classical biochemical techniques has become impractical.

Multiplex technologies, particularly bead-based platforms, have addressed the challenge of increasing throughput without compromising sensitivity or accuracy and can be applied in all aspects of the drug discovery process, from target identification through post-clinical analysis. Revealed here are data from multiplex protein detection experiments that shed new light on protein function and pathological significance. These data are both consistent with results obtained by traditional detection methods and represent savings in time and sample.

Researchers today are all too aware of the complex interactions among proteins, among signaling pathways, and within protein complexes. As a result, all aspects of drug discovery and development face the same rate-limiting bottleneck: quantification of multiple proteins that, together, define particular cellular conditions or responses to drugs.

Historically, scientists have quantified protein levels using enzyme-linked immunosorbent assays (ELISAs), and have detected protein sizes and post-translational modifications using Western blotting. Though these methods are considered the “gold standards” of protein characterization, measuring proteins one at a time makes it difficult to derive biologically meaningful or statistically significant results from experiments that perturb entire cells, tissues, or organisms. Typically, researchers need large amounts of sample, time, and assay reagents to perform ELISAs or Western blots for all the proteins relevant in an experiment.

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Figure 2A: Receptor tyrosine kinase- (RTK) and stress-mediated signaling result in activation of MAPK and SAPK pathways in A431 cells. Diagram shows which phospho-proteins were detected by either multiplex bead-based detection (Panel B) or Western blotting (Panel C). (Source: David Hayes, Millipore)

Figure 2B: A431 cells were cultured until approximately 80% confluent. Control and treated cultures were serum-starved for four hours prior to treatment. Culture media was then changed to treatment medium containing either 100 ng/mL EGF (stimulated), or an equivalent amount of vehicle control (unstimulated). The cells were stimulated for five minutes, then washed with ice-cold PBS. Cells were lysed using MILLIPLEX map Lysis Buffer (Millipore) with protease inhibitors. 10 ?g total protein was used for immunoprecipitation. The lysate sample was incubated overnight with Luminex assay beads. The beads were washed the next morning, and SDS-PAGE was performed on equivalent amounts of stimulated and unstimulated sample. Gels were transferred to nitrocellulose, and blotted with the appropriate biotinylated primary antibody. Results demonstrate the specificity of the beads and the appropriate assay response to the stimulation conditions used for each lysate. (Source: David Hayes, Millipore)

Figure 2C: Lysates were prepared as above and 10 ?g total protein was loaded into each well containing MAPK Pathway 10-plex assay beads. Samples were assayed in triplicate. Unstimulated HeLa cell lysate was used as a universal negative assay control for all targets in most MILLIPLEX map kits. The signal from unstimulated HeLa cells was previously determined to be identical to the background signal from unstimulated A431 cells (data not shown). Samples were assayed according to kit instructions, and results are given as the average value and standard deviation of the median fluorescence intensities obtained from the Luminex 200 instrument. (Source: David Hayes, Millipore)

Multiplexing—detecting many molecular species simultaneously from a single sample—overcomes these limitations, which researchers face at every step of modern drug discovery. Figure 1 illustrates the application of multiplex protein detection in six stages of drug discovery and development. The workflow begins with target identification, in which biomarkers—a set of proteins that characterize a healthy or disease state—are measured in subjects to generate a list of possible drug targets. At the end of the drug development process, biomarker levels in clinical trial subjects also indicate the efficacy of a drug, and can confirm its mechanism. Intermediate stages, such as profiling the toxicity of panels of drug candidates, also benefit from multiplex protein detection.

As early as 1977, scientists outlined methods to use bead-conjugated antibodies for multiplex detection using common multipurpose flow cytometers.¹ However, multiplex technology came into widespread use in the mid-1990s, with the development of commercial bead-based array kits, specialized sorting/detection instruments, and integrated software programs that automatically process huge data sets.

A new gold standard?
Scientists consider protein detection by ELISA to be the most reliable method of measuring protein levels in biological samples. The ELISA uses an antibody “sandwich,” in which soluble protein is bound by a primary antibody that is conjugated to a solid surface. A second, soluble “reporter” antibody, typically labeled with a fluorescent or chemiluminescent tag, recognizes the bound protein, and the signal intensity correlates to the amount of bound target protein.

Bead-based protein detection, in which the primary antibody is bound to a solid bead and recognizes soluble target protein, is similar to ELISA in that proteins are measured via immunodetection. However, as in any multiplexing experiment, there is the potential for cross-reactivity between antibodies and epitopes, introducing possible artifacts. Several studies have compared protein quantification results between ELISA and bead-based multiplex assays, and these studies have been meta-analyzed in a review comparing different commercial bead-based multiplex assay kits.²

Although several different multiplex platforms are available, such as the Cytometric Bead Array (BD Biosciences, San Jose, Calif.) and FlowCytomix (Beckman Coulter, Fullerton, Calif.), most of the analyses comparing multiplex bead-based quantification to ELISA have been performed using Luminex xMAP technology (Luminex Corporation, Austin, Texas), the first flow cytometry platform designed specifically for multiplex bead analysis. The results of these comparative studies show remarkable agreement with ELISA, with correlation coefficients consistently above 0.90.²

Luminex xMAP technology consists of beads containing two different fluorescent dyes at varying concentrations, creating 100 different fluorescent “signatures” of beads. Each bead can then be conjugated to a different antibody, theoretically allowing the simultaneous detection of 100 distinct analytes.³ Luminex makes xMAP beads commercially available to allow companies and end users, to create their own custom antibody arrays. The high throughput format of the assay kits makes internal quality control and standard curve generation easier than with ELISA. Consequently, increasing numbers of drug discovery and development researchers are using the Luminex xMAP multiplex assay instead of ELISA for protein quantification.²

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Figure 3. Validation of a human ovarian cancer multiplex biomarker kit by comparison with ELISA. Serum samples from healthy patients and those diagnosed with ovarian cancer were tested for the indicated proteins with the MILLIPLEX map Human Cancer Biomarker 6-Plex Panel and the results recorded as serum concentrations.⁵ Comparison of these data (top panel) with ELISA results (bottom panel) indicate that both methods of biomarker detection show the same trends in comparing healthy and diseased patients.⁵ (Source: Reprinted with permission from Visintin, I. et al. Diagnostic markers for early detection of ovarian cancer. Clinical Cancer Research. 2008;14(4):1065-1072, Supplementary Figure 1)

Cellular responses to stimuli used to be depicted as linear chains of events with a single starting point leading to a single outcome. The current view of the cell instead shows multiple, parallel responses to stimuli communicating in intersecting networks of signals, with divergent downstream responses. Multiplexing has allowed simultaneous determination of multiple biomarkers in a single cellular state, so researchers can construct a complex pathway with a single experiment.

To illustrate the utility of multiplexing in pathway analysis, researchers used a multiplex cell signaling assay kit, the MILLIPLEX map Human MAPK/SAPK – 10 Plex (Millipore, Billerica, Mass.), to determine the proteins that are phosphorylated in response to epidermal growth factor (EGF). Figure 2 shows both the mitogen-activated protein kinase (MAPK) pathway and stress-associated protein kinase (SAPK) pathway activated by EGF in A431 human epithelial carcinoma cells, which express high levels of EGF receptor. Out of a panel of 10 downstream kinase substrates, five were found to be activated by EGF by analyzing lysates in a single multiplex Luminex assay. Western blotting using the same phospho-specific antibodies used in the multiplex assay confirmed the results.

Measuring multiple biomarkers to define healthy or diseased states is more reliable than trying to correlate levels of individual molecules to disease progression. Visintin and colleagues, after establishing the first set of biomarkers for a blood test for ovarian cancer, recently adapted the assay using a multiplex bead-based cancer biomarker detection kit, the MILLIPLEX map Human Cancer Biomarker 6-Plex Panel from Millipore.^4,5 Although more research is required before the kit can be used commercially for early diagnosis, comparison of the multiplex assay results with ELISA in Figure 3 shows that both methods have the same trends in leptin, osteopontin, prolactin, and IGF-II levels in healthy and diseased patients.

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Figure 4. Workflow comparison between multiplex bead-based protein detection and ELISA. “Internal control” is defined as a protein with known or constant concentration that is present in the same sample/well as the variable protein of interest. (Source: David Hayes, Millipore)

Multiplex vs. ELISA workflows
Besides saving the researcher time and sample usage, bead-based multiplex protein detection can improve data quality because internal controls can be measured in the same well, with increased dynamic range of detection. Figure 4 summarizes the similarities and differences in workflows between Luminex xMAP Technology and classic ELISA. Both methods are equally sensitive; however, for analyzing multiple proteins, multiplexing provides more results in less time.

In addition to cell signaling proteins, cytokines—including lymphokines, interferons, colony-stimulating factors, and chemokines—were some of the first biomarkers to be measured in multiplex bead-based assays. Specialized subsets of cytokines, in combination with other biomarkers, are now available as kits to study other therapeutic areas, including neurobiology, ophthalmology, and bone metabolism. The ability of cytokines and their receptors to modulate immune response events has piqued interest in their use as immunotherapeutic agents. Widening use of multiplex bead-based assays in clinical settings is expected, as suppliers design kits that are compatible with more sample types.

References
1. Horan PK, Wheeless LL. Quantitative single cell analysis and sorting. Science. 1977;198:149-157.
2. Eishal, MF, McCoy, JP. Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods. 2006;38(4):317-323.
3. https://www.luminexcorp.com/01_xMAPTechnology, accessed January 19, 2009.
4. Mor, G. et al. Serum protein markers for early detection of ovarian cancer. PNAS. 2005; 102(21):7677-7682.
5. Visintin, I. et al. Diagnostic markers for early detection of ovarian cancer. Clinical Cancer Research. 2008;14(4):1065-1072.

About the Author
David Hayes’s experience includes kinase and protease profiling at GlaxoSmithKline and Luminex assay development for Upstate Biotechnology. He leads research and development of cell signaling and disease biomarkers at Millipore Corporation.

This article was published in Drug Discovery & Development magazine: Vol. 12, No. 3, March, 2009, pp. 22-25.