Genetic Engineering & Biotechnology News

OCT15 2017

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Genetic Engineering & Biotechnology News | GENengnews.com | OCTOBER 15, 2017 | 17 bound protein. Beads are then re-suspended in a solution of streptavidin-β-galactosidase (SBG) for 10 minutes and washed twice. The plate containing the prepped beads is then loaded into the SR-Plex for imaging and analysis. In the SR-Plex, an automated pipet- ting module uses disposable pipette tips to automatically mix the beads with a solution of resorufin β-d-galactopyranoside (RGP), a fluorogenic substrate of SBG. The mixture is transferred into microfluidic arrays on a Si- moa disc, where each sample is loaded into an individual array containing 216,000 fem- toliter-sized wells, which have been sized to hold no more than one bead per well (4.25 µ m width, 3.25 µ m depth). After allowing the beads to settle into the wells, the SR-Plex adds a volume of oil to seal each well of the array, creating a confined volume of 40 fL. If the target molecule is present (that is, if an immunocomplex has formed), then the captured enzyme label will convert the RGP substrate into a fluorescent product. Since the Simoa disc separates the paramagnetic beads into small volumes, each array con- tains a high local concentration, enabling the platform to detect accumulated fluorescent RGP products catalyzed by a single enzyme molecule. The SR-Plex finally captures images of each array on the Simoa disc using LED- based illumination and a 12-bit scientific grade CMOS camera. The camera acquires two fluorescence images of the RGP signal emitted from each array, which enables the SR-Plex software to measure an increase in signal and thereby confirm the presence of a true immunocomplex. It can also distinguish between beads that are associated with a single enzyme molecule (an "on" well) and beads that are not associated with an enzyme (an "off" well). The SR-Plex image analysis software determines the average enzyme per bead (AEB) for both calibrators and samples, and it interpolates the sample concentrations from the calibration curves using its curve- fitting feature. Improving Inflammatory Biomarker Detection with Multiplexing Using the SR-Plex, the new Human Cytokine 6-Plex Panel 1 can simultane- ously detect and quantify up to six protein biomarkers associated with inflammation and immunotherapy, including biomarkers IL-10, IL-12p70, IL-17A, IL-6, TNFα, and IFNγ, from a single sample. In one case, sci- entists used this panel to detect and quantify these biomarkers at endogenous levels from healthy donors. 5 The assay's high sensitiv- ity enabled it to detect all six markers in all samples at sub-pg levels (Figure 2). It also exhibited a very low level of cross-reactivity among the biomarkers (Table). The Simoa Human Cytokine 6-Plex Panel 1 is the first commercial immunoassay capable of simul- taneously detecting these critical inflamma- tory biomarkers in serum and plasma at normal levels, providing researchers with a new tool for investigating the role of in- flammation in disease progression and the response to immunotherapy. Conclusions The SR-Plex with Simoa technology al- lows researchers to detect and quantify previously undetectable proteins, serum, plasma, CSF, or other sample types. This technology creates the potential to detect biomarkers of disease, such as cancer and inflammatory conditions, much earlier. The newly designed SR-Plex benchtop instru- ment is an economical option for high-sen- sitivity biomarker analysis, and may one day yield the discovery of biomarkers with applications across many different fields of clinical research. www.Trianni.com ©TRIANNI, Inc 7 TRIANNI's antibody discovery platform, The Trianni Mouse, represents best-in-class technology for effi cient generation of fully-human monoclonal antibodies. The Trianni Mouse provides a complete heavy, kappa and lambda repertoire in a single organism. Variable domain exon sequences are human while the genetic machinery that drives antibody expression, including extensively optimized promoters and enhancers, is of mouse origin. This Mouse off ers biologic drug researchers a facile solution for isolation of exceptional therapeutic antibody candidates. For more information email info@trianni.com. Exceptional Human Antibody Discovery OMICS Tutorial Dandan Shan, Ph.D. (dshan@quanterix. com), is principal scientist, assay development, and David Wilson, Ph.D. (dwilson@quanterix.com), is senior director, product development, at Quanterix. Website: www.quanterix.com. References available online. Figure 2. Six inflammatory biomarkers (6 from plasma and 6 from serum for a total of 12 data points) were detected and quantified from 20 healthy donors. Error bars represent median and interquartile ranges. Table. This table shows the high specificity and low level of cross- reactivity between the six biomarkers in the Human Cytokine 6-Plex Panel 1 assay. There was very little cross-talk among the six analytes in the assay.

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