Sponsored By: Custom Publishing From: BIOMARKER DISCOVERY: AN ARRAY OF POTENTIAL Page 3 Page 4 Page 5 Page 6 Candidate Screening: Journey of a A Multiplexed Overcoming Low Abundance with Biomarker Candidate Approach Bottlenecks in High Sensitivity Cancer Treatment BROADEN YOUR PERSPECTIVE See up to 2000 proteins at a glance. The RayBio® L2000 Array Our largest antibody array to date: includes 2000 human proteins on your choice of glass slide or membrane support. Explore an extensive panel of metabolic enzymes, structural proteins, epigenetic markers, hormones, and neuroregulatory factors – in addition to our popular list of cytokines, growth factors, receptors, adipokines, proteases, and signaling proteins. Ideal for biomarker discovery studies and exploratory screens. Available as an easy-to-use kit or a full service. Get more out of your sample. RayBiotech Learn more R Empowering your proteomics 1.888.494.8555 / RAYBIOTECH.COM / ISO 13485:2016 BIOMARKER DISCOVERY: AN ARRAY OF POTENTIAL Candidate Screening: Low Abundance with High Sensitivity hen processes regulating life – such as growth, of mass spectrometry instruments. Large-panel arrays allow reproduction, and movement – go awry, disease researchers with modest budgets to screen for hundreds Wcan result. Chemical moieties become imbalanced of potential biomarkers, including cytokines, chemokines, in these disease states, often resulting in measurable levels of and other secreted molecules that could be missed through certain signature molecules, termed biomarkers. Biomarker traditional proteomics approaches. discovery has traditionally been performed through A Streamlined Workflow: From Discovery to proteomic approaches. However, these techniques have been limited in their ability to detect low abundance molecules. Validation Moreover, they are generally unable to scan for chemokines, Once thought of as being too narrow in scope for the biomarker cytokines, growth factors, and other secreted molecules. discovery phase, arrays containing a thousand or more Antibody arrays can overcome many of these issues and antibodies are now routinely used, enabling the researcher have become a more utilized proteomics approach in recent to target a broad selection of candidate markers with known years, thanks largely to intensified efforts to test and validate importance in many key cellular pathways and diseases. Thus, antibody pairs, facilitating the development of broad, high- antibody arrays may be used either alone or in tandem with throughput screens. MS data to perform the initial biomarker screen. The array offers an additional advantage: the ability to streamline the Complementing Traditional Biomarker Discovery biomarker workflow, from discovery to validation, by making Techniques use of common antibodies across multiple assays. Traditional proteomic methods, such as 2-D gel MS-based biomarker verification usually involves a targeted electrophoresis and mass spectrometry (MS, including liquid approach, such as multiple-reaction-monitoring (MRM). chromatography-MS; LC-MS), have become indispensable Array-based biomarker verification instead makes use of tools in biomarker discovery. These techniques have smaller quantitative arrays (multiplex ELISAs) containing particularly advanced cancer biomarker discovery. The a panel of analytes identified by the initial screening array. unbiased nature of mass spectrometry-based screens makes Because smaller antibody arrays are relatively quick to them ideal techniques for mining protein targets that don't produce, and the antibodies don’t need to be developed from have antibodies or other affinity reagents available. In recent scratch, the verification stage can begin quickly. years, significant efforts have been made to improve MS-based approaches in an effort to overcome associated biomarker- The final step in the biomarker journey, validation, requires discovery challenges (e.g., the need to perform high- assessment of biomarker performance in a large cohort study throughput assays and the wide dynamic range of biomarker in the target population. Array-based technology again allows candidate concentrations in the human body)1. this stage to be simplified, permitting the use of the same antibodies employed in the verification stage. However, due to ion suppression, certain secreted and low abundance biomarkers can be missed using MS- or 2-D gel- Sensitive and High-Throughput techniques, and different approaches and equipment are required to fully mine the proteome for these biomarkers. Furthermore, Multiplex immunoassays offer high detection sensitivity (as the cost of equipment, need for highly trained personnel, low as 1 pg/ml). Furthermore, antibody arrays are adaptable and high costs per sample tested can be prohibitive to many to high-throughput analysis, and the choice of array platforms researchers wanting to use traditional proteomic methods. offered is large and always expanding. The development of high-density arrays has placed the technique securely into the High-density antibody array screening, that is, using antibody spotlight when it comes to antibody discovery and validation, panels with more than 200 markers, requires minimal training making antibody arrays a perfect complement to traditional to use; moreover, capital equipment requirements (microarray proteomic techniques. scanners or CCD cameras) represent a fraction of the cost For references, please see page 7. TheScientist 2019 3 the-scientist.com JOURNEY OF A BIOMARKER CANDIDATE Potential biomarkers for a particular disease or condition must first be discovered: high-density screening antibody arrays are an excellent tool for this stage, allowing researchers to profile hundreds of disease-related proteins simultaneously with minimal material and cost. For biomarker candidate validation and verification, multiplex antibody arrays or single-target ELISAs are a logical choice: there is no need to develop new antibodies or immunoassays for validation or clinical applications using an antibody array approach. HIGH-DENSITY SCREENING ARRAY • Phosphoproteins ANTIGENS FROM PATIENT SERA • Glycoproteins • Proteins (Antigens) candidate identification Multiplex antibody array • Quantitative clinical Application • Diagnose and monitor disease • Potential therapeutic target candidate Validation candidate Verification • No need to develop new assay BIOMARKER DISCOVERY: AN ARRAY OF POTENTIAL A Multiplexed Approach ore and more studies are finding that a single (antigens), glycoproteins, phosphoproteins, and many other cytokine or other chemical moiety is insufficient moieties. With the number of validated antibodies growing every Mfor use as a true disease biomarker, and that a more year, the gap in target throughput between arrays and mass global perspective is needed to truly understand the presence spectrometry continues to narrow. Antibody arrays are designed or development, of disease. High-density multiplexed with a pre-selected panel of antibodies that target markers antibody arrays that can now contain 2,000 antibodies or having known or suspected relevance in many disease processes. more in a single panel, combined with an ever-increasing Thus, this targeted approach can efficiently identify proteins pool of validated antibodies, puts arrays at the forefront of that are more likely to be effective biomarkers. Furthermore, biomarker discovery and development, and makes finding their high sensitivity makes them ideal for identifying secreted multiple biomarkers more accessible than ever before. molecules that may, in combination with other moieties, provide the global perspective necessary to tailor therapeutic regimes. Evolution of the Antibody Array Arrays in Precision Medicine Microarrays were first conceptualized in the early 1980s by Dr. Tse Wen Chang, who demonstrated that 20x20 grids of For precision medicine, antibody arrays are proving to be antibody spots could be placed on a small surface1. Antibody fundamental for the diagnosis and treatment of disease. For array models were further developed by Dr. Roger Ekins and example, high-density arrays were used to better characterize colleagues in the late 1980s when they created a model that the expression levels of 200 human cytokines, leading to the permitted simultaneous screening of an analyte panel2. Initial identification of a common cytokine signature and guiding concepts tried to miniaturize immunological assays and were the diagnosis of a patient with idiopathic uveitis. Personalized normally performed in 96-well plates. However, glass slides and treatment reversed the vision loss, illustrating how arrays may membranes were soon found to be better substrates for placing assist in individualizing therapy4. Using a similar approach, minute antibody spots, as they could accommodate larger arrays. another study identified serum proteins that could potentially identify patients who would respond to ipilimumab, a targeted In late 2000, the idea of simultaneously detecting multiple melanoma therapy5. cytokines came to fruition in the mind of Dr. Ruo-Pan Huang at Emory University, who developed nitrocellulose membrane- Uncovering New Drug Targets based antibody arrays as a strategy to increase the efficiency of profiling blood proteins. Dr. Huang began developing antibody Antibody arrays, with their high-throughput multiplexed design, arrays to replace cumbersome and expensive biochemical are a tool for biomarker discovery and not only offer insight into techniques such as single-target ELISAs and Western