Genetic Engineering & Biotechnology News

OCT15 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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16 | OCTOBER 15, 2017 | | Genetic Engineering & Biotechnology News Dandan Shan, Ph.D., and David Wilson, Ph.D. The future of immuno-oncology treatment and autoimmune diagnostics will involve the detection and quantification of inflammato- ry biomarkers. As immunotherapy becomes a more common approach for treating can- cer and inflammatory disease, cytokines will not only play a role in determining a patient's disease diagnosis, prognosis, and potential treatment options, but also may serve as a primary index of a therapy's effectiveness. Incorporating these biomarkers into clini- cal practice, however, has been a challenge. Only a few cytokines have been studied in relation to inflammatory disease and immu- notherapy, mainly because the biomarkers for these conditions are typically present in very small concentrations, and current research methods aren't sensitive enough to detect them. It is critical that physicians have the ability to quantify these cytokines to track dis- ease progression before and after treatment. This tutorial explains how to use the Human Cytokine 6-Plex Panel 1 with the new benchtop Quanterix SR-Plex™ Ultra- Sensitive Biomarker Detection System to simultaneously detect and quantify six in- flammatory biomarkers with unprecedent- ed sensitivity using an ELISA-based meth- od. This advanced approach reduces assay volume requirements for multiple sample types and may be sensitive enough to de- tect circulating disease biomarkers before symptoms appear. As a result, it could potentially provide physicians with a new capability to track disease progression, and the effectiveness of treatments against diseases, which they previously could not monitor with such precision. Ultra-Sensitive Biomarker Technology The SR-Plex utilizes Simoa™ (Single Mol- ecule Array) technology, a digital ELISA ap- proach that functions through the same basic mechanisms as traditional ELISAs—but it is 1,000 times more sensitive, which allows it to detect cytokines in the bodily fluids of both healthy and ill individuals. 1 The technology derives its sensitivity from dividing the ELISA reaction into tens of thousands of 40-femto- liter-sized wells and measuring the presence of the target molecule separately in each one with single-molecule precision (Figure 1). In a study from the Journal of Immuno- logical Methods, Simoa technology was proven useful for monitoring therapeutic efficacy—it was the first technique to suc- cessfully measure changes in the concentra- tion of circulating Crohn's disease cytokines following the administration of a specific biological therapeutic. 2 Recently, a proof-of- concept study demonstrated applications for this technology in drug-induced liver toxic- ity. Specifically, researchers were able to rap- idly diagnose sepsis in patients and perform pharmacokinetic measurements of interfer- ing nucleic acid therapeutics. 3 Digital Method Detects Previously Undetectable Proteins Simoa technology starts with building an immunocomplex using paramagnetic beads as the solid phase. 4 These beads are labeled with fluorescent dyes of various wavelengths and concentrations to create optically dis- tinct subpopulations, and antibodies to spe- cific proteins are immobilized to them. Mix- tures of these beads are pooled together to create a multiplex assay. After they are pooled, the beads are combined with a mixture of biotinylated detection antibodies and added to a 100-µL sample of prediluted sample fluid (typically a 1:4 dilution of blood, urine, or cerebral spi- nal fluid) in a 96-well plate for a 30-minute incubation. 720,000 beads are added to each sample, resulting in a high bead-to-molecule ratio. The high bead concentration yields two primary advantages: 1. The percentage of beads that contain a labeled immunocomplex follows a Pois- son distribution—at low concentrations of protein, the Poisson distribution indicates that each bead will capture either a single immunocomplex or none. For example, if 60,000 protein molecules are captured and labeled on 500,000 beads, then 12% (60,000/500,000) of the beads will carry one protein molecule, and 88% will not carry any protein molecules. 2. With so many beads in solution, the bead-to-bead distance is small, so every molecule encounters a bead in less than a minute. At this time scale, the target analyte molecules, and even large proteins, diffuse quickly. In theory, all the molecules should have multiple collisions with multiple beads. Next, using an automated plate washer, the magnetic beads are pelleted to remove un- Improve Detection Limits and Preserve Sample Volumes with High-Sensitivity Multiplexing Assays Examining Inflammatory Cytokines in Serum and Plasma OMICS Insights Genomics & Proteomics Cost-effective sequencing can now provide insight into disease prevention and intervention. Though the tools for sequencing are readily available, resources for interpreting results and helping patients under- stand the information have been less accessible. How- ever, a new partnership between Fabric Genomics and Genome Medical is looking to provide expert medical interpretation and counseling to physicians and patients on genomic data. Genome Medical will use Fabric Genomics' clinical interpretation platform, Fabric Enterprise ™ , for ongoing examination of its pa- tients' genomic data. "By combining our expertise in genome interpreta- tion with Genome Medical's team of physicians and genetic counselors, we can offer patients and physi- cians a clinical solution by combining genomic knowl- edge with patient care to improve their health," ex- plained Martin Reese, Ph.D., CEO at Fabric Genomics. Through this partnership, Genome Medical's ex- pert team of physicians and genetic counselors will be able to turn to Fabric Enterprise to augment the information provided to individuals and clinicians. Genome Medical's workflow will now provide an end- to-end offering by helping patients and physicians decide whether they need a test, how to order the right test, how to interpret test results, understand ways to covert the results into actionable medical de- cisions, and reinterpret DNA over the course of treat- ment, if applicable. "Year after year, our knowledge about how genetics affects health and our ability to apply this knowledge to improve patient health grows dramatically," stated Steven Bleyl, M.D., Ph.D., CMO at Genome Medical. "We believe genomic medicine is a lifelong journey. To that end, it will be increasingly important that our clinicians have the right tools to view, query, interpret and reinterpret our patients' genetic information over time. We have partnered with Fabric Genomics to meet that crucial need." n A Partnership for Improved Understanding of Genetic Results Tutorial Figure 1. Paramagnetic Simoa beads are used as the solid phase in a sandwich immunoassay containing an enzymatic label. The labeled beads are mixed with a fluorgenic substrate and loaded into an array of femtoliter-size wells on the Simoa disc, where the beads settle into individual wells. The wells are sealed with a layer of oil, which prevents the beads from diffusing out of the wells and allows the fluorescent enzyme product to accumulate at high concentrations, enabling detection of a single enzyme molecule. Fluorescence imaging and data reduction is used to calculate the concentration of each analyte in the assay.

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