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ELISPOT ADDRESSES the COMPLEX QUESTIONS ARISING from VACCINE and NOVEL THERAPIES Posted by Rolando Pajon, Phd

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ELISPOT ADDRESSES the COMPLEX QUESTIONS ARISING from VACCINE and NOVEL THERAPIES Posted by Rolando Pajon, Phd ELISPOT ADDRESSES THE COMPLEX QUESTIONS ARISING FROM VACCINE AND NOVEL THERAPIES Posted by Rolando Pajon, PhD The accelerated arrival of novel vaccines and immunotherapies into the clinical space spurred the emergence of fields like personalized medicine, immuno-profiling and immuno-monitoring built around increasingly sophisticated testing platforms. Among them, immunoassays in the ELISpot (Enzyme-Linked ImmunoSpot) family are the most frequently used functional assays for single-cell analysis.1 Using 2017 data from Trialtrove (Citeline.com), we found that ELISpot assays were used in more than 160 open clinical trials (Figure 1). The main drivers for this rising clinical usage are: ▶ The increased prevalence of infectious and chronic diseases as the population ages in developed countries ▶ Extensive use of immunoassays in oncology new vaccines and immunotherapies ▶ Technological developments, such as test automation and rapid analysis ▶ Growth in the biotechnology sector Looking for the Right Spot In the ELISpot assay, cell-secreted analytes (cytokine, immunoglobulin, etc.) are captured by an antibody- coated membrane at the bottom of ELISA-like plates. After removal of cells, the captured analyte is visualized by deposition of a colored substrate, which results in the formation of spots on the membrane. Spots are a representation of cells that secrete the analyte of interest, the amount of analyte secreted and its kinetics.2 The spots are then counted and quantitative reports are generated like the frequency of antigen-specific T-cells3 following immunization or treatment.4 The ELISpot assay where human interferon gamma is detected is the IFN-g ELISpot assay. IFN-g ELISpot The induction of a potent lymphocyte effector function is central to the activity of novel biological therapeutics. Due to its known role, IFN-g is a predominant marker for the induction of cellular immune responses.5-7 Correspondingly, for the past two decades, the IFN-g ELISpot assay has become a highly sensitive yet “simple” platform to detect and quantify antigen-specific cellular responses. It has also become the benchmark for analysis of T-cell responses according to Good Clinical Laboratory Practice guidance.8 It is used in vaccines, transplantation, HIV, cancer and allergy, 4,5,6,7 and more. The IFN-g ELISpot assay carries nearly 55% of all ELISpot testing needs in ongoing clinical trial evaluation. Because of that, and the ongoing industry expansion into polyfunctional cell analysis,9,10 much attention has been given to the quality of the IFN-g ELISpot assay. However, the simplicity of the IFN-g ELISpot assay only seems apparent. Results from proficiency panels initiated by several organizations revealed drastic differences in the reported results by participant labs.11,12,13 There are several strictly controlled variables that influence the assay outcome. Those include the test medium, the antigen (stimulus), cell storage conditions, cell viability, staff training levels and the actual counting and quantification approach of the ELISpot plates. A Holistic Approach to ELISpot Development, Qualification and Validation The successful implementation of any ELISpot testing solution during clinical trials does not happen in a vacuum if assay variables and sample integrity are to be kept in check. Part of the Covance IFN-g ELISpot integrated solution is a dedicated Targeted Cell Isolation team, who is responsible for processing/producing the cellular component that goes into the assay (peripheral blood mononuclear cells, T-cells, B-cells, etc.), avoiding costly sample processing, minimizing risk and additional fees incurred when shipping live frozen cells to different specialty laboratories. Likewise, regulatory proficiency, global reach, proven logistics and a dedicated, scalable ELISpot solution with increasing testing volumes – according to the specific study needs – are all critical to the success of a clinical trial program using ELISpot. Covance, at its Central Laboratories (CLS), offers a wide array of tests in the areas of genomics, flow cytometry and histology – all under one roof as these tests generate mutually complementary data to that of ELISpot assays. This enables sponsors to create functional immunoprofiles that capture the overall cellular immunity status of clinical trial subjects. Covance has offered IFN-g ELISpot testing for several years in our Translational Biomarker Solutions laboratories. However, and more importantly, Covance just added IFN-g ELISpot capabilities at our Vaccine and Novel Immunotherapeutic Laboratory Solutions (within the CLS) in Indianapolis, offering sponsors a one-stop solution for any IFN-g ELISpot assay in their clinical testing programs. Positioned for the Future In addition to the traditional colorimetric quantification of secreted cytokines, the Covance ELISpot platform allows for the development and execution of multiplexed FluoroSpot assays (Figure 2), the next- generation of ELISpot, which enables simultaneous measurement of different analytes.14,15 The capability to validate novel FluoroSpot assays using a modular approach will allow for a “plug-and-play” flexible offering that will decrease development time for sponsor-specific antigens and targets. The ELISpot platform at Covance’s Vaccine and Novel Immunotherapeutic Laboratory department is ideally positioned to support upcoming developments in the field. References 1- Czerkinsky CC, et al J Immunol Methods. 1983;65:109-121. 2- Karulin A.Y, Lehmann, PV. Methods Mol Biol. 2012;792:125-143. 3- Nanan R, et al J Gen Virol. 2000;81:1313-1319. 4- Carvalho L.H, et al. J Immunol Methods. 2001;252:207-218. 5- Flynn K., et al. Immunity. 1998;8:683-691. 6- Bercovici N, et al. Clin Diag Lab Immunol. 2000;7:859-864. 7- Carter LL, Swain SL. Curr Opin Immunol. 1997;9:177-182. 8- Ezzelle J, et al. J Pharm Biomed Anal. 2008;46(1):18-29. 9- Dillenbeck T, et al. Cells. 2014;3(4):1116-1130. 10- Gazagne A, et al. J Immunol Methods. 2003;283:91-98. 11- Janetzki S, et al. Cancer Immunol. Immunother. 2008;57:303-315. 12- Britten C.M, et al. Cancer Immunol Immunother. 2008;57:289-302. 13- Cox J.H, et al. AIDS Res Hum Retroviruses. 2005;21:68-81. 14- Janetzki S, et al. Cells. 2014;3(4):1102-1115. 15- Gazagne A, et al. J Immunol Methods. 2003;283(1-2):91-98. Learn more about our drug development solutions at www.covance.com Covance Inc., headquartered in Princeton, NJ, is the drug development business of Laboratory Corporation of America® Holdings (LabCorp®). Covance is the marketing name for Covance Inc. and its subsidiaries around the world. The Americas + 1.888.COVANCE + 1.609.452.4440 Europe / Africa + 00.800.2682.2682 Asia Pacific + 800.6568.3000 © Copyright 2017 Covance Inc. ARTCLS039-1017.
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