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Multiplexing technology has come a long way since ELISA. Researchers could not be happier.  

Given the high cost and financial risk involved in drug development, scientists are continually looking for tools that effectively demonstrate a candidate drug’s efficacy and ensure minimal toxicity or other adverse effects. Merck drew world-wide attention in 2004 with the withdrawal of VIOXX (rofecoxib) due to the increased risk of serious thrombotic cardiovascular adverse events. While the resulting litigation and public scrutiny have provoked much debate over drug safety in the United States, it is clear that drug developers need better tools that will identify safety concerns early in the development pipeline, thus preventing significant economic impact and potential morbidity and mortality in those patients treated with new drugs.

Drug companies have begun investing significant resources towards identification of biomarkers—characteristics that can be objectively measured and evaluated as an indicator of normal biologic or pathogenic processes or pharmacological responses to a therapeutic intervention.

A search of the PubMed database for the term “biomarker” will return several hundred journal articles published in the last 30 days. Using tools for both genomics and proteomics, scientists are routinely identifying new biomarkers for conditions such as cancer, heart disease, and tissue transplantation. Many drug companies now have biomarker discovery programs that run in parallel to their drug development programs. If relevant biomarkers are identified, this approach will save significant time and resources by eliminating poor drug candidates at the early stages of drug development while pushing ideal drug candidates further into clinical development.

Just as PCR and DNA sequencing have revolutionized research into the human genome, recent advancements in DNA microarray technology have pushed the limits of genomics even further, thus allowing the simultaneous detection of thousands of genes. Along similar lines, the field of proteomics has been equally impacted by the development of protein microarrays that allow the simultaneous detection of multiple protein targets in biological samples, or examination of protein-protein interactions, protein kinase activity, or targets of biologically active small molecules. By coating solid substrates bearing defined patterned arrays or microspheres containing defined fluorescent signatures with known detection molecules (nucleic acids probes, antibodies, or other proteins), scientists now have more powerful tools to further define host factors associated with human disease.

As with any advancement, there are limitations. While multiplex protein analysis has become a popular new tool for analyzing antibodies, cytokines, allergens, drugs, and hormones, this technology presents several challenges to the clinical lab such as quality control and availability of diagnostic algorithms to transform the raw data into diagnostic results.1 Yet, scientists continually seek those tools that expedite the generation of larger amounts of data from a single nucleic acid sample, biological specimen, or cell lysate. The utility of multiplex measurements lies not only in the ability to screen individual marker candidates, but also in the ability to evaluate multiple markers in combination.2 Thus, while steps are taken to ensure the reliability of protein multiplexing in clinical diagnostics, this technology has already allowed researchers to begin discovering new biomarkers and therapeutic strategies for autoimmune disease, genetic and infectious disease, and cancer. For example, use of protein microarrays to screen sera from patients with autoimmune diseases will facilitate the identification of autoantibody signatures that can be used for diagnosis and, potentially, the prognosis of patients.2 In the field of cancer, microarrays have been used to identify biological subsets of disease that have prognostic relevance and thus improve the ability to predict whether a cancer will have a significant response to therapy.3 These studies suggest that introduction of this information into clinical trials will lead to more biologically-based stratification schemes and the development of more tailored therapies.3 

ELISA
In the 1970s, the development of the ELISA technique and monoclonal antibodies revolutionized science by providing immunologists with a convenient method to detect the presence of an antibody or antigen in a biological sample. The ELISA soon became a powerful research tool that, with an ever-expanding repertoire of monoclonal antibodies against a wide range of host and pathogen proteins, gives researchers the ability to search for biomarkers specific to a specific immune or disease process. However, a significant limitation with an ELISA is its ability to measure only one analyte at a time; thus, in order to measure multiple analytes in one biological sample, there must be a large enough volume of the sample to divide among multiple ELISA plates.

While a wide range of ELISA kits are commercially available and convenient for use by laboratories with standard 96-well plate readers, samples that are divided
Calibration curves 
click to enlarge
 
Figure 1. Calibration curves for IL-6, IL-1ß, TNF-a, and IL-8 (left to right) from a calibrator preparation diluted serially in the MSD assay (4-plex human cytokine panel)
 
among these kits are susceptible to sampling errors since each kit requires different sample dilutions and dilution buffers that can influence inter-assay variability. The development of protein multiplexing has overcome this limitation by complexing detection antibodies in patterned arrays on solid substrates or multiplexed bead systems.4 With the ability to analyze multiple analytes in one small sample volume, variability is minimized since the analytes are diluted into the same dilution buffer. This allows better comparisons between analytes since they are measured in the same sample.

In the mid-1990s, particle-based flow cytometric assays developed by Luminex Corporation (Austin, Texas) revolutionized protein multiplexing by using microspheres as the solid support for conventional immunoassays.4 In simple terms, a specific detection antibody was coupled to a microsphere with a unique fluorescent signature. By mixing sets of these microspheres coated with different detection antibodies, researchers used flow cytometry to detect up to 100 analytes within the same sample. In recent years, the technology has been expanded beyond protein expression profiling into the areas of gene expression profiling and genotyping, as well as diagnostics (allergy testing, autoimmune disease, HLA testing, and infectious disease).

As a contract research organization, Southern Research Institute continually looks for technologies that facilitate the increased speed of data collection and analysis for both its commercial and government clients. In 2004, researchers in the Infectious Disease Research department of the Southern Research Institute, Birmingham, Ala. and Frederick, Md., began evaluating the technologies for protein multiplexing. This effort was initiated in order to maximize the amount of data that could be acquired from pre-clinical drug studies performed in tissue culture (primary cells and cell lines) or murine small animal models. Among the technologies evaluated was a multiplexing technology developed by Meso Scale Discovery (MSD; Gaithersburg, Md.). This technology uses a carbon electrode microplate (MULTI-SPOT plate) containing patterned arrays of detection antibodies and electrochemiluminescence detection with labels that emit light upon electrochemical stimulation at the electrode surfaces. As a result, this platform offers increased sensitivity, a wide dynamic range, and minimal background since only signal close to the surface of the microplate is detected by the instrument reader (Figure 1).

Like other technologies, the MSD system offers an assortment of cytokine kits that allow the ability to multiplex standard mouse and human cytokine profiles. The technologies under review offered good sensitivity and dynamic range, which varied depending on the cytokines being tested. Overall, the researchers found that the MSD platform offered two advantages: overall convenience of use and minimal background signal.

Compared to other systems, the MSD SECTOR Imager machine is virtually maintenance-free and can read a 96-well plate in a fraction of the time of competing devices (~1 minute/plate vs. >30 minutes/plate for other technologies). Also, since only those labels near the electrode surface of the plate are detected, many assays do not require a wash step. This can significantly reduce the amount of technical time required to process a plate, enabling the processing of more plates and thus more samples.

Depending on the sample being assayed, it is often important to minimize the background signal of the assay. High background can impact the sensitivity of the assay and study results. While this may not be a factor when determining cytokine levels in culture supernatants or in plasma/serum, it can be critical when evaluating cytokines in certain biological samples. For example, samples from the mucosa can have high levels of mucin and proteases that negatively impact background levels in standard ELISAs. If limited sample dilution is required, these samples may also impact microsphere aggregation and sample uptake in Luminex-based systems. Researchers found that overall, samples tested in the MSD system were much less susceptible to high background issues compared to the same sample types tested using the standard ELISA format or Luminex.

Southern Research Institute has used MSD electrochemiluminescence technology to evaluate cytokines in serum/plasma from mice, nonhuman primates, and humans, as well as culture supernatants from cell lines and peripheral blood mononuclear cells. By combining this technology with standard in vitro and in vivo assays to evaluate a drug’s antiviral activity or ability to modulate immune function, Southern Research can now optimize the amount of data generated in a particular study by increasing the speed of data collection and analysis. More recently, given the straightforward nature of assay development on this platform, scientists at Southern Research Institute have begun developing improved assays to complement available commercial kits. Just as monoclonal antibodies helped expand the utility of the standard ELISA, the next generation of multiplexing is only as limited as the detection reagents that are currently available. Scientists now have at their disposal improved multiplexing tools that, by identifying new biomarkers or combinations of biomarkers, will lead to the development of new drugs, as well as novel diagnostic tests,5 predictive panels for the diagnosis, staging, and monitoring of cancer or other diseases,6 and prognostic markers of clinical outcome such as predictive markers of response to immune therapies.7

About the Author
Dr. Cummins has over 15 years experience in the field of mucosal immunology. He is actively involved in the pre-clinical development of topical microbicides and currently manages a range of projects that include use of in vitro assays and animal models for the evaluation of candidate anti-viral drugs.

Literature Cited
1. Master, S.R., C. Bierl, and L.J. Kricka. Diagnostic challenges for multiplexed protein microarrays. Drug Discov Today., 2006. 11(21-22): p. 1007-11. Epub 2006 Sep 26.
2. Cekaite, L., E. Hovig, and M. Sioud. Protein arrays: a versatile toolbox for target identification and monitoring of patient immune responses. Methods Mol Biol., 2007. 360: p. 335-48.
3. Sears, C. and S.A. Armstrong. Microarrays to identify new therapeutic strategies for cancer. Adv Cancer Res., 2007. 96: p. 51-74.
4. Vignali, D.A., Multiplexed particle-based flow cytometric assays. J Immunol Methods., 2000. 243(1-2): p. 243-55.
5. Harwanegg, C. and R. Hiller. Protein microarrays for the diagnosis of allergic diseases: state-of-the-art and future development. Allerg Immunol (Paris). 2006. 38(7): p. 232-6.
6. Dehqanzada, Z.A., et al. Assessing serum cytokine profiles in breast cancer patients receiving a HER2/neu vaccine using Luminex technology. Oncol Rep., 2007. 17(3): p. 687-94.
7. Yurkovetsky, Z.R. et al., Multiplex Analysis of Serum Cytokines in Melanoma Patients Treated with Interferon-{alpha}2b. Clin Cancer Res., 2007. 13(8): p. 2422-8.

This article was published in G & P magazine: Vol. 7, No. 10, October, 2007, pp. G2-G4.

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