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Assay Guidance Manual

Edited by G. Sitta Sittampalam, PhD, Editor-in-chief, Neely Gal-Edd, MS, Associate Managing Editor, Michelle Arkin, PhD, Douglas Auld, PhD, Chris Austin, MD, Bruce Bejcek, PhD, Marcie Glicksman, PhD, James Inglese, PhD, Vance Lemmon, PhD, Zhuyin Li, PhD, James McGee, PhD, Owen McManus, PhD, Lisa Minor, PhD, Andrew Napper, PhD, Terry Riss, PhD, O. Joseph Trask, Jr., and Jeff Weidner, PhD.

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Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.

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The collection of chapters in this eBook is written to provide guidance to investigators who are interested in developing assays useful for the evaluation of collections of molecules to identify probes that modulate the activity of biological targets, pathways, and cellular phenotypes. These probes may be candidates for further optimization and investigation in drug discovery and development.

Originally written as a guide for therapeutic project teams within a major pharmaceutical company, this manual has been adapted to provide guidelines for scientists in academic, non-profit, government and industrial research laboratories to develop potential assay formats compatible with High Throughput Screening (HTS) and Structure Activity Relationship (SAR) measurements of new and known molecular entities. Topics addressed in this manual include:

Development of optimal assay reagents.
Optimization of assay protocols with respect to sensitivity, dynamic range, signal intensity and stability.
Adopting screening assays from bench scale assays to automation and scale up in microtiter plate formats.
Statistical concepts and tools for validation of assay performance parameters.
Secondary follow up assay development for chemical probe validation and SAR refinement.
Data standards to be followed in reporting screening and SAR assay results.
Glossaries and definitions.

This manual will be continuously updated with contributions from experienced scientists from multiple disciplines working in drug discovery & development worldwide. An open submission and review process will be implemented in the near future on this eBook website, hosted by the National Library of Medicine with content management by the National Center for Advancing Translational Sciences (NCATS, http://ncats.nih.gov/), the newest component of the National Institutes of Health (NIH).

Preface
Introduction
- Basics of Assay Equipment and Instrumentation for High Throughput Screening
  G. Sitta Sittampalam.
  Published May 1, 2012.
- HPLC-MS/MS for Hit Generation
  Stefan Jon Thibodeaux, David A Yurek, and James E McGee.
  Published March 1, 2013.
- Early Drug Discovery and Development Guidelines: For Academic Researchers, Collaborators, and Start-up Companies
  Michael Hughes, James Inglese, Andrew Kurtz, Ali Andalibi, Lavonne Patton, Chris Austin, Michael Baltezor, Michael Beckloff, Sitta Sittampalam, Michael Weingarten, and Scott Weir.
  Published May 1, 2012; Last Update: October 1, 2012.
BioAssay Ontology
- Development and Applications of the Bioassay Ontology (BAO) to Describe and Categorize High-Throughput Assays
  Uma D. Vempati and Stephan C. Schürer.
  Published May 1, 2012; Last Update: October 1, 2012.
Cell-Based Assays
- FLIPR™ Assays for GPCR and Ion Channel Targets
  Michelle R. Arkin, Patrick R. Connor, Renee Emkey, Kim E. Garbison, Beverly A. Heinz, Todd R. Wiernicki, Paul A. Johnston, Ramani A. Kandasamy, Nancy B. Rankl, and Sitta Sittampalam.
  Published May 1, 2012; Last Update: October 1, 2012.
- Cell-Based RNAi Assay Development for HTS
  Kok Long Ang, Farhana Feroze, Gopinath Ganji, Yue Li, Jeffrey Weidner, and Sitta Sittampalam.
  Published May 1, 2012; Last Update: October 1, 2012.
- Assay Development Guidelines for Image-Based High Content Screening, High Content Analysis and High Content Imaging
  William Buchser, Mark Collins, Tina Garyantes, Rajarshi Guha, Steven Haney, Vance Lemmon, Zhuyin Li, and O. Joseph Trask Jr.
  Published October 1, 2012.
- Advanced Assay Development Guidelines for Image-Based High Content Screening and Analysis
  Mark-Anthony Bray, Anne Carpenter, and Imaging Platform, Broad Institute of MIT and Harvard.
  Published March 1, 2013.
- Nuclear Factor Kappa B (NF-κB) Translocation Assay Development and Validation for High Content Screening
  O. Joseph Trask Jr.
  Published October 1, 2012.
- Ion Channel Screening
  Owen B McManus, Maria L Garcia, David Weaver, Melanie Bryant, Steven Titus, and James B Herrington.
  Published October 1, 2012.
Biochemical Assays
- Basics of Enzymatic Assays for HTS
  Harold B. Brooks, Sandaruwan Geeganage, Steven D. Kahl, Chahrzad Montrose, Sitta Sittampalam, Michelle C. Smith, and Jeffrey R. Weidner.
  Published May 1, 2012; Last Update: October 1, 2012.
- Validating Identity, Mass Purity and Enzymatic Purity of Enzyme Preparations
  John E Scott, Kevin P Williams, Tod Holler, and Andrew Napper.
  Published May 1, 2012; Last Update: October 1, 2012.
- Mechanism of Action assays for Enzymes
  John Strelow, Walthere Dewe, Phillip W Iversen, Harold B Brooks, Jeffrey A Radding, James McGee, and Jeffrey Weidner.
  Published May 1, 2012; Last Update: October 1, 2012.
- Protease Assays
  Guofeng Zhang, Tod Holler, and Andrew Napper.
  Published May 1, 2012; Last Update: October 1, 2012.
- Assay Development for Protein Kinase Enzymes
  J. Fraser Glickman, James McGee, and Andrew Napper.
  Published May 1, 2012; Last Update: October 1, 2012.
- Inhibition of Protein-Protein Interactions: Non-Cellular Assay Formats
  Michelle R. Arkin, Marcie A. Glicksman, Haian Fu, Jonathan J. Havel, and Yuhong Du.
  Published March 18, 2012; Last Update: October 1, 2012.
- Receptor Binding Assays for HTS and Drug Discovery
  Douglas S Auld, Mark W. Farmen, Steven D. Kahl, Aidas Kriauciunas, Kevin L. McKnight, Chahrzad Montrose, and Jeffrey R. Weidner.
  Published May 1, 2012; Last Update: October 1, 2012.
- GTPγS Binding Assays
  Neil W. DeLapp, Wendy H. Gough, Steven D. Kahl, Amy C. Porter, and Todd R. Wiernicki.
  Published May 1, 2012; Last Update: October 1, 2012.
- Phospho-ERK Assays
  Kim E. Garbison, Beverly A. Heinz, Mary E. Lajiness, Jeffrey R. Weidner, and G. Sitta Sittampalam.
  Published May 1, 2012.
- Impedance-Based Technologies
  Kim E. Garbison, Beverly A. Heinz, Mary E. Lajiness, Jeffrey R. Weidner, and G. Sitta Sittampalam.
  Published May 1, 2012.
- IP-3/IP-1 Assays
  Kim E. Garbison, Beverly A. Heinz, and Mary E. Lajiness.
  Published May 1, 2012.
- Immunoassay Methods
  Karen L. Cox, Viswanath Devanarayan, Aidas Kriauciunas, Joseph Manetta, Chahrzad Montrose, and Sitta Sittampalam.
  Published May 1, 2012; Last Update: October 1, 2012.
Validation
- HTS Assay Validation
  Philip W. Iversen, Benoit Beck, Yun-Fei Chen, Walthere Dere, Viswanath Devanarayan, Brian J Eastwood, Mark W. Farmen, Stephen J. Iturria, Chahrzad Montrose, Roger A. Moore, Jeffrey R. Weidner, and G. Sitta Sittampalam.
  Published May 1, 2012; Last Update: October 1, 2012.
- Assay Operations for SAR Support
  Benoit Beck, Yun-Fei Chen, Walthere Dere, Viswanath Devanarayan, Brian J. Eastwood, Mark W. Farmen, Stephen J. Iturria, Phillip W. Iversen, Steven D. Kahl, Roger A. Moore, Barry D. Sawyer, and Jeffrey Weidner.
  Published May 1, 2012; Last Update: October 1, 2012.
- Calculations and Instrumentation used for Radioligand Binding Assays
  Steven D. Kahl, G. Sitta Sittampalam, and Jeffrey Weidner.
  Published May 1, 2012; Last Update: October 1, 2012.
Data and Compound Management
- Data Standardization for Results Management
  Robert M. Campbell, Julia Dymshitz, Brian J. Eastwood, Renee Emkey, David P. Greenen, Julia M. Heerding, Dwayne Johnson, Thomas H. Large, Thomas Littlejohn, Chahrzad Montrose, Suzanne E. Nutter, Barry D. Sawyer, Sandra K. Sigmund, Martin Smith, Jeffrey R. Weidner, and Richard W. Zink.
  Published May 1, 2012; Last Update: October 1, 2012.
In Vivo Assays
- In Vivo Assay Guidelines
  Joseph Haas, Jason Manro, Harlan Shannon, Wes Anderson, Joe Brozinick, Arunava Chakravartty, Mark Chambers, Jian Du, Brian Eastwood, Joe Heuer, Stephen Iturria, Philip Iversen, Dwayne Johnson, Kirk Johnson, Michael O’Neill, Hui-Rong Qian, Dana Sindelar, and Kjell Svensson.
  Published May 1, 2012; Last Update: October 1, 2012.
Glossary
- Glossary of Quantitative Biology Terms
  Viswanath Devanarayan, Barry D. Sawyer, Chahrzad Montrose, Dwayne Johnson, David P. Greenen, Sitta Sittampalam, Terry Riss, and Lisa Minor.
  Published May 1, 2012.

Cover photo courtesy of Manju Swaroop, Research Scientist, NCATS

All Assay Guidance Manual content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported license (CC BY-NC-SA 3.0), which permits copying, distribution, transmission, and adaptation of the work, provided the original work is properly cited and not used for commercial purposes. Any altered, transformed, or adapted form of the work may only be distributed under the same or similar license to this one.

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