Genetic Engineering & Biotechnology News

SEP15 2017

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10 | SEPTEMBER 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News GEN: What is the biggest consider- ation researchers have when deciding between multiplex immunoassays and standard assays? Dr. Pregibon: For many researchers, the de- cision to multiplex is driven by the desire to obtain a breadth of biological data from a limited sample volume. In addition, as the field better understands how proteomics, ge- nomics, and epigenetics can be used together to describe complex biological states both in vitro and in vivo, the assessment of several biomarkers across many types of targets can provide a more comprehensive snapshot of a disease and accelerate research outcomes. With only 50 µL of serum, it is possible for scientists to use Abcam's FirePlex ® platform to profile 10–20 proteins or 20–50 microR- NAs, and do this over hundreds of samples. Dr. Tan: Prior to embarking on an effort to develop or validate multiplex biomarker as- says, one should critically evaluate whether candidate assays suffice to enable one to go beyond the conventional "one target, one drug" approach. ''Composite profiling'' may not be the only rationalization for using mul- tiplex assays. The justification should be driv- en by the need to probe biological networks, dissect pathways, and monitor physiological and pathological states. Oftentimes, this key consideration is influenced by the complexity of developing the multiplex assays (such as biological expression levels) and the fear of added expense to validate them. Ms. Johnson: I think this can vary by research project. If sensitivity is the most important fac- tor, or if only one or two proteins need to be measured, then a standard assay is an excel- lent solution, like Bio-Techne's Quantikine® ELISA kits. However, if sample is precious and/or a researcher is in the discovery stage of a project, then multiplex immunoassays can be a better choice. For up to four analytes, Simple Plex™ Assays afford researchers the best of both worlds: multianalyte measure- ment with single-analyte performance. Dr. Wild: Of course, the price is an important aspect and if only a few analytes need to be ex- amined, standard assays would likely be the first choice. The analysis of several analytes in parallel within a single assay, however, reduces handling steps and thereby improves the robust- ness and comparability of results. Additionally, usually smaller amounts of starting material are required, making a multiplex approach very at- tractive for rare samples. In my point of view, proving the absence of analytes can also pro- vide important information. In some cases, one might even detect something unexpected just by looking for more than the usual suspects. Dr. Mao: From my point of view, the cost, sample volume, availability of analytes, and target detection sensitivity are the four most important aspects to consider. For biomarker discovery, researchers often prefer "big nets" to find meaningful targets. We address this need by offering the world's largest sandwich- based, quantitative antibody array, capable of measuring over a thousand human proteins in one experiment, at a cost of around a dol- lar per target. The cost may be reduced even further by adopting the single-antibody, di- rect antigen-labeling approach. This method doesn't permit quantitative output, but does give relative signal changes. Dr. Chimento: The biggest considerations when deciding between the multiplex and standard assays are availability of suitable multiplex and platform equipment to run the assay along with the availability of the test samples. Developing a singleplex or standard assay is straightfor- ward, as buffers and other reagents can be easily designed to detect a single protein target. Multiplex assays have an inherently lon- ger development time as the optimization issues are compounded. These frequently make use of a single multianalyte sample in a specific diluent, which may not be opti- mal for all the components in the multiplex. Multiplex assays, however, may be critically important when the test samples are limiting, allowing researchers to collect required data on an otherwise limiting sample. GEN: What are some of the main challenges companies face when developing multiplex immunoassays? Dr. Pregibon: The development of multiplex immunoassays is predominantly plagued by unwanted interactions and interference between antibodies, targets, off-targets, ma- trices, and substrates. A great immunoassay starts with great antibodies and ends with validation across relevant biological matrices to combat these issues. Abcam's monoclonal rabbit antibodies are high-affinity, high-spec- ificity binders, and our FirePlex immunoas- says, which are built on bio-inert substrates, are validated across several biological matri- ces to ensure experimental compatibility. Dr. Tan: The entire scaffold of antibody screening for the development of multiplex immunoassays depends critically on the pair- ing of high-quality capture and detection an- tibodies. The main challenge of working with these reagents is dealing with variability, such as between-lot and between-vendor variabil- ity. This can be extremely difficult to delineate empirically during the assay development process, in which the variability in these re- agents is buried deep and magnified in a mul- tiplex setting. We take great care to minimize the effects of this variability in our assays. Managing assay cross-talk is another chal- lenge in developing these assays. Even with top- quality antibodies, nonspecific binding arises when many different antibodies are blended for multiplex testing. We carefully select and screen antibodies in the multiplex environment to choose the best pairs for each particular assay. Ms. Johnson: The biggest challenge is bal- ancing quality vs. quantity. Bio-Techne's goal is to multiplex as many analytes as pos- sible without sacrificing accuracy and preci- sion within the assay. While developing our Human XL Cytokine Discovery Luminex ® Performance Assay, we spent a great deal of time optimizing assay diluents. We have a large supply of antibodies at our disposal, but the raw ingredient require- ments for each analyte can be vastly differ- ent. Inevitably, analytes must be left out be- cause they don't meet our strict performance specifications. Researchers should be wary of large multiplex assays that promise single- analyte assay performance. Dr. Wild: Finding the right capture reagents (such as antibodies) is important for every assay, but it becomes crucial for multiplex assays. Of course, the antibody must be spe- cific for its target, but it must also work in the context of a multiplex platform. In addition, unwanted interactions be- tween the antibodies, detection reagents, and analytes should be anticipated. If such interactions are possible, they must be sub- ject to detection. Quality control of the com- ponents as well as for the complete assay are very important and must be carefully devel- oped in parallel to the assay itself. Dr. Mao: The development of multiplex im- munoassays poses the following challenges: • Availability of high-quality antibodies: Analyte detection by sandwich antibody pairs must be validated by both calibration curves and relevant biological samples. • Cross-reactivity between antibodies: For sandwich-based immunoassays, the combinations of paired antibodies within an array panel can occasionally cross- react. Before assembling the panel, rigorous cross-reactivity testing must be performed for each antibody pair with every other antibody pair. The larger the panel, the more laborious array development becomes. Roundup Continued from page 8 Drug Discovery Insights Discovery & Development If organ-on-a-chip technology gives you brainwaves or makes your heart beat faster, you may want to check on its progress every so often. After all, as Roche Pharmaceutical Science's Adrian Roth cau- tioned in a Nature news article a couple of years ago, inflated expectations for the technology are to be avoided. They could lead to disillusionment, particu- larly among pharma companies. If pharma were to decide that organ-on-chip applications were not, in fact, imminent, they could create a self-fulfilling prophecy, even in the face of optimistic market pro- jections. (According to Research and Markets, the global organ-on-a-chip market is expected to grow at approximately 69.4% over the next decade to reach approximately $6.13 billion by 2025.) For what it's worth, organs-on-chips have been entering the mainstream of news, if not the main- stream of disease modeling, drug screening, or patient stratification. For example, back in April 2017, the FDA announced that it intended to use organ-on-a-chip technology to study the effects of potential chemical and biological hazards found in foods, cosmetics, or dietary supplements. The FDA added that the chips by Emulate—which were originally developed for drug testing—might work better than cell-culture or animal- based tests, at least for the agency's purposes. Emulate also indicated that organ chips were get- ting a boost from the space program. The company announced in June 2017 that it had received a grant to modify its human Brain-Chip system and create an experimental platform that could be used aboard the International Space Station. More recent developments include news from TissUse (which licensed ProBioGen's human lymph node technology for multi-organ-chip applications); CN Bio Innovations (which licensed a Bristol-Myers Squibb hepatitis B program to enhance its liver-on-a-chip model); and Hurel (which announced the commercial launch of its microliver testing platform). An even more recent development: Mimetas, in collaboration with Roche, announced that it had devel- oped a novel gut-on-a-chip system that could be used to evaluate epithelial barrier function and predict ad- verse drug effects. The system was described in Nature Communications, in an article entitled, "Membrane- Free Culture and Real-Time Barrier Integrity Assess- ment of Perfused Intestinal Epithelium Tubes." Accord- ing to Mimetas, the gut model "lends itself to assess toxicology and transport of oncological and other compounds, as well as disease-modeling studies and fundamental research." n Drug Development in the (Organ) Chips

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