This eBook is a comprehensive, crucial resource for investigators optimizing assays to evaluate collections of molecules with the overall goal of developing probes that modulate the activity of biological targets, pathways or cellular phenotypes. Such probes might 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 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:

• Descriptions of assay formats that are compatible with HTS and determination of SAR
• Selection and development of optimal assay reagents
• Optimizations and troubleshooting for assay protocols with respect to sensitivity, dynamic range, signal intensity and stability
• Adaptations of assays for automation and scaling to microtiter plate formats
• Instrumentation
• Sources of assay artifacts and interferences
• Statistical validation of assay performance parameters
• Secondary assays for chemical probe validation and SAR refinement
• Data standards for reporting the results of screening and SAR assays
• In vivo assay development and validation
• Assay development and validation for siRNA-based high-throughput screens

The National Center for Advancing Translational Sciences (NCATS) manages the content of the Assay Guidance Manual with input from industry, academia and government experts. More than 100 authors from around the globe have contributed content to this free resource, which is updated quarterly with contributions by experienced scientists from multiple disciplines working in drug discovery and development worldwide.

For more information about the Assay Guidance Manual and related training opportunities, visit https://ncats.nih.gov/expertise/preclinical/agm.

Contents

• New in Assay Guidance Manual
• Preface
  G. Sitta Sittampalam and Nathan P. Coussens.
  Published May 1, 2012; Last Update: March 31, 2017.
• Acknowledgements
• Considerations for Early Phase Drug Discovery
  Editors: Thomas D.Y. Chung, Nathan P. Coussens, G. Sitta Sittampalam, and Yueming Wang.
  • Early Drug Discovery and Development Guidelines: For Academic Researchers, Collaborators, and Start-up Companies
    Jeffrey Strovel, Sitta Sittampalam, Nathan P. Coussens, Michael Hughes, James Inglese, Andrew Kurtz, Ali Andalibi, Lavonne Patton, Chris Austin, Michael Baltezor, Michael Beckloff, Michael Weingarten, and Scott Weir.
    Published May 1, 2012; Last Update: July 1, 2016.
    • Abstract
    • Background
    • Purpose
    • Scope
    • Assumptions
    • Definitions
    • Section 1. Discovery and Development of New Chemical Entities
    • Section 2. Repurposing of Marketed Drugs
    • Section 3. Development of Drug Delivery Platform Technology
    • Section 4. Alternative NCE Strategy: Exploratory IND
    • Section 5. Orphan Drug Designation
    • Conclusion
    • References
• In Vitro Biochemical Assays
  Editors: Nathan P. Coussens, Timothy L. Foley, Sam R.J. Hoare, James McGee, and G. Sitta Sittampalam.
  • Analyzing Kinetic Binding Data
    Sam R.J. Hoare.
    Published April 1, 2021.
    • Abstract
    • Introduction
    • Basic Principles of Ligand Binding Kinetics
    • Kinetic Analysis Methods for Direct Target-Ligand Binding Assays
    • Competition Kinetics: Quantifying Kinetics by Competition Against a Tracer Ligand
    • Compound Washout Method
    • Complex Binding Mechanisms
    • Binding Kinetics from Functional Assays
    • Summary
    • Appendix: Equations for Binding Kinetic Data Analysis
    • References
  • Validating Identity, Mass Purity and Enzymatic Purity of Enzyme Preparations
    John E Scott and Kevin P Williams.
    Published May 1, 2012; Last Update: October 1, 2012.
    • Abstract
    • Definitions
    • Consequences of using Enzymatically Impure Enzyme Preparations.
    • Signs of Enzymatic Contamination
    • Solutions for Enzymatic Contamination
    • Importance of Batch Testing
    • Identity and Mass Purity
    • Assay Design Factors that Affect the Likelihood of Detecting Enzyme Impurities
    • Validating Enzymatic Purity
    • References
  • 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.
    • Abstract
    • Enzyme Assay Development Flow Chart
    • Introduction
    • Concept
    • Reagents and Method Development
    • Detection System Linearity
    • Enzyme Reaction Progress Curve
    • Measurement of K_m and V_max
    • Determination of IC₅₀ for Inhibitors
    • IC₅₀ Determination for SAR
    • Optimization Experiments
    • Assay Validation
    • References
  • 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.
    • Abstract
    • Overview of MOA in Drug Discovery
    • Types of Inhibition
    • Performing MOA Studies
    • References
    • Glossary of MOA Terms
  • Assay Development for Protein Kinase Enzymes
    J. Fraser Glickman.
    Published May 1, 2012; Last Update: October 1, 2012.
    • Abstract
    • Introduction
    • Assay Development
    • Considerations of Mechanism
    • Assay Optimization
    • Acknowledgements
    • References
    • Additional References:
  • 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: July 1, 2018.
    • Abstract
    • Introduction
    • Flow Chart of Steps to Assay Development for SPA Format
    • Flow Chart of Steps to Assay Development for Filter Format
    • Scintillation Proximity Assays (SPA)
    • SPA Assay Format
    • Assay Buffer
    • Solvent Interference Conditions
    • Binding Parameter
    • Filtration Assays
    • Filter Assay Format
    • Assay Buffer (Filter)
    • Assay Conditions (Filter)
    • Binding Parameters (Filter)
    • Practical Use of Fluorescence Polarization in Competitive Receptor Binding Assays
    • References
  • Protease Assays
    Guofeng Zhang.
    Published May 1, 2012; Last Update: October 1, 2012.
    • Abstract
    • Introduction
    • Homogenous Assays
    • Separation-Based Assays
    • Triaging Assay Hits
    • Data Analysis for Time Dependent Inhibitors and Covalent Inhibitors
    • Conclusion
    • References
  • 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.
    • Abstract
    • Overview and Introduction
    • ELISA-type assays
    • Mix-and-read assays
    • Validating drug-like binding of PPI inhibitors
    • Useful websites
    • References
    • Additional References
  • Immunoassay Methods
    Karen L. Cox, Viswanath Devanarayan, Aidas Kriauciunas, Joseph Manetta, Chahrzad Montrose, and Sitta Sittampalam.
    Published May 1, 2012; Last Update: July 8, 2019.
    • Abstract
    • Immunoassay Development, Optimization and Validation Flow Chart
    • Introduction
    • Immunoassay Parameters
    • Reagents
    • Instrumentation
    • Immunoassay Formats
    • Important Parameters for Development of an Immunoassay
    • Initial Concept and Method Development for a Sandwich Immunoassay
    • Initial Concept and Method Development of a Competitive Assay
    • Development of a Competitive Immunoassay
    • Method Validation (Pre-Study)
    • Method Validation (In-Study)
    • Pre-Study & In-Study Acceptance Criteria
    • Precision Profile Tool for Estimating Assay Performance Characteristics
    • Immunogenicity Cut Point Analysis Tool (I-CAT)
    • References
    • Additional References
  • 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.
    • Abstract
    • Flow Chart for Assay Development
    • Introduction
    • Materials and Reagents
    • Membrane Preparations and Assay Buffers
    • Basic Assay Protocol
    • Assay Optimization
    • Data Analysis
    • Filtration Assays
    • Non-Radiometric GTPγS Assays
    • References
    • Additional References
  • Histone Acetyltransferase Assays in Drug and Chemical Probe Discovery
    Sanket Gadhia, Jonathan H. Shrimp, Jordan L. Meier, James E. McGee, and Jayme L. Dahlin.
    Published July 26, 2017; Last Update: July 1, 2018.
    • Abstract
    • Flowchart
    • Abbreviations
    • Introduction and Background
    • General Considerations for HAT Assays
    • Biochemical HAT Assays
    • Section Summary
    • Cell-Based HAT Assays
    • Miscellaneous Considerations
    • Conclusions
    • Acknowledgements
    • Conflict of Interest Statement
    • Suggesting Readings (alphabetical order)
    • References
• In Vitro Cell Based Assays
  Editors: Marcie Glicksman, Zhuyin Li, Sarine Markossian, Terry Riss, G. Sitta Sittampalam, O. Joseph Trask, Jr., and Yueming Wang.
  • Machine Learning and Assay Development for Image-based Phenotypic Profiling of Drug Treatments
    Jonathan Z. Sexton, Reid Fursmidt, Matthew J. O’Meara, Wienand Omta, Arvind Rao, David A. Egan, and Steven A. Haney.
    Published March 15, 2023.
    • Abstract
    • Flow Chart
    • Introduction
    • Methods and Resources
    • Protocol Overview
    • Image Analysis: Getting from Images to Numbers
    • Exploratory Data Analysis in Pilot Screening
    • Machine Learning for a Group of Plates
    • Machine Learning, Incorporating Novel Phenotype Identification, With Applications to Cell Painting and Related Phenotypic Screens
    • Conclusions
    • References
  • Biosensor Assays for Measuring the Kinetics of G-Protein and Arrestin-Mediated Signaling in Live Cells
    Sam R.J. Hoare and Thomas E. Hughes.
    Published September 1, 2021.
    • Abstract
    • Introduction
    • Measuring GPCR Signaling Using Genetically-Encoded Fluorescent Biosensors
    • Analyzing Signaling Time Course Data
    • Simplifying Biased Agonism Assessment Using Signaling Kinetics
    • Conclusions
    • Acknowledgements
    • References
  • Cell Viability Assays
    Terry L Riss, Richard A Moravec, Andrew L Niles, Sarah Duellman, Hélène A Benink, Tracy J Worzella, and Lisa Minor.
    Published May 1, 2013; Last Update: July 1, 2016.
    • Abstract
    • Introduction
    • Conclusion
    • References
  • Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells
    Terry Riss, Andrew Niles, Rich Moravec, Natashia Karassina, and Jolanta Vidugiriene.
    Published May 1, 2019.
    • Abstract
    • Introduction
    • Dyes That Selectively Penetrate Dead Cells
    • Fluorescent DNA Binding Dyes That Penetrate Dead Cells
    • Optional Protocol to Estimate Total Number of Cells
    • Markers That Leak Out of the Cytoplasm of Dead Cells into Culture Medium
    • Conclusion
    • References
  • Apoptosis Marker Assays for HTS
    Terry L. Riss, Martha A. O’Brien, Richard A. Moravec, Kevin Kupcho, and Andrew L. Niles.
    Published July 1, 2021.
    • Abstract
    • Abbreviations
    • Introduction
    • Caspase Activity Detection
    • Protocol to Detect Caspase-3/7 Activity as a Marker of Apoptosis
    • Detection of PS Exposure as a Marker of Apoptosis
    • Protocol to Detect PS Exposure as a Marker of Apoptosis
    • Data Calculation
    • Commercial Reagents
    • Conclusion
    • References
  • Pluripotent Stem Cell Culture Scale-Out
    Debra L. Johnson, Robert Scott, and Rhonda Newman.
    Published June 1, 2021.
    • Abstract
    • Overview and Flowchart of Assay Guidelines
    • Introduction
    • Stem Cell Culture Considerations
    • Assay Protocol: Scale-out of hPSCs in Monolayer Culture Conditions
    • Monitoring Cell Health in Large-Scale Culture
    • Conclusion
    • References
  • Authentication of Human and Mouse Cell Lines by Short Tandem Repeat (STR) DNA Genotype Analysis
    Jamie L. Almeida and Christopher T. Korch.
    Published January 17, 2023.
    • Abstract
    • Introduction
    • Authentication of Mouse Samples by STR Analysis
    • STR DNA genotyping assays for authentication of human and mouse cell line identities and detection of cross-contaminating intraspecies cells
    • Data Analysis and Interpretation
    • Criteria for Determining Quality STR Profile Analysis for Reliable and Interpretable Results
    • Services for STR Genotyping of Cell Lines
    • Troubleshooting
    • Non-commercial Kits and Inappropriate Authentication Methods
    • Glossary of Terms
    • License
    • Acknowledgements
    • Useful Resources
    • References
  • In vitro 3D Spheroids and Microtissues: ATP-based Cell Viability and Toxicity Assays
    Monika Kijanska and Jens Kelm.
    Published January 21, 2016.
    • Abstract
    • Flow Chart: 3D Spheroid and Microtissue Growth and Assay Development
    • Introduction
    • 1. In Vitro Toxicity and Drug Efficacy Testing in a 3D Spheroid Model
    • 2. 3D Microtissue Viability Assay
    • References
  • Cell-Based RNAi Assay Development for HTS
    Scott Martin, Gene Buehler, Kok Long Ang, Farhana Feroze, Gopinath Ganji, and Yue Li.
    Published May 1, 2012; Last Update: May 1, 2013.
    • Abstract
    • Introduction
    • Off-Target Effects
    • Loss-of-Function Screens Using siRNA
    • siRNA Hit Selection
    • RNAi Synthetic Lethality Screens
    • Loss-of-Function Screens Using shRNA
    • Appendix
    • Acknowledgements
    • Abbreviations
    • References
  • 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.
    • Abstract
    • Overview: FLIPR™ Assay Development
    • Introduction
    • Types of FLIPR™ Formats
    • Reagents and Buffers for Method Development
    • Method Development and Optimization
    • FLIPR Instrument Setup
    • Potential Artifacts
    • References
  • Ion Channel Screening
    Owen B McManus, Maria L Garcia, David Weaver, Melanie Bryant, Steven Titus, and James B Herrington.
    Published October 1, 2012.
    • Abstract
    • 1. Introduction
    • 2. Fluorescence Assays Using Membrane Potential Sensing Dyes
    • 3. Ion Flux Assays
    • 4. HTS Assay Considerations
    • 5. Preparation of Cells for Automated Electrophysiology
    • References
  • Automated Electrophysiology Assays
    Birgit T. Priest, Rok Cerne, Michael J. Krambis, William A. Schmalhofer, Mark Wakulchik, Benjamin Wilenkin, and Kevin D. Burris.
    Published March 9, 2017.
    • Introduction
    • Flowchart
    • Choice of Instrumentation
    • Choice of Cell Line
    • Choice of Solutions
    • High-Throughput Automated Electrophysiology Assays
    • Medium-Throughput Automated Electrophysiology Assays
    • Low-Throughput Automated Electrophysiology Assays
    • Cost of Operation
    • References
    • Appendix 1: Protocol for Assessment of Use-Dependent Sodium Channel Inhibitors on IonWorks Quattro
    • Appendix 2: Protocol for Assessment of KCNQ1/KCNE1 Currents on IonWorks Barracuda
    • Appendix 3: Protocol for Assessment of GABA_A Current Block on IonWorks Barracuda
    • Appendix 4: Protocol for Assessment of State-Dependent Sodium Channel Inhibitors on PatchXpress
  • 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.
    Published October 1, 2012; Last Update: September 22, 2014.
    • Abstract
    • 1. Introduction
    • 2. Image Technologies and Instruments
    • 3. Assay Concept and Design
    • 4. Cellular Models for High Content Experiments
    • 5. Assay Development Considerations and TroubleShooting
    • 6. Image Acquisition, Analysis and Data Interpretation
    • 7. Assay Validation for HCA Screens
    • 8. Data Management for High Content Screening
    • 9. References
  • Advanced Assay Development Guidelines for Image-Based High Content Screening and Analysis
    Mark-Anthony Bray, Anne Carpenter; Imaging Platform, Broad Institute of MIT and Harvard.
    Published July 8, 2017.
    • Abstract
    • 1. Experimental design for HCS
    • 2. Assay Quality AND Acceptance Criteria for HCS
    • 3. Quality Control for HCS
    • 4. Normalization of HCS data
    • 5. Measurement of image features
    • 6. Machine learning for HCS
    • 7. Whole-organism HCS
    • 8. Acknowledgements
    • 9. References
  • Nuclear Factor Kappa B (NF-κB) Translocation Assay Development and Validation for High Content Screening
    O. Joseph Trask, Jr.
    Published October 1, 2012.
    • Abstract
    • Introduction and background
    • Assay Development Considerations
    • Assay Development
    • Reference Compounds
    • Plate Layout
    • Protocol for Activators (Agonist) of NFκB Pathway
    • Logistics Analysis of Protocol
    • Cell Based Screen
    • Screening Commercially Available NF-κB Antibodies (Appendix 1)
    • Safety Considerations: Guidelines & Precautions (Appendix 2)
    • Acknowledgements
    • References
  • High Content Screening with Primary Neurons
    Hassan Al-Ali, Murray Blackmore, John L Bixby, and Vance P. Lemmon.
    Published October 15, 2013; Last Update: October 1, 2014.
    • Abstract
    • 1. Introduction
    • 2. Prelminary Assay considerations
    • 3. Assay Development
    • 4. Additional Considerations
    • 5. Protocols for HCS with Primary Neurons
    • Acknowledgments
    • References
  • Phospho-ERK Assays
    Authors: Kim E. Garbison, Beverly A. Heinz, Mary E. Lajiness, Jeffrey R. Weidner, and G. Sitta Sittampalam.
    Published May 1, 2012; Last Revision: June 25, 2015.
    • Abstract
    • Introduction
    • Overview of Technology
    • AlphaScreen SureFire ERK Assay
    • Characteristics of the AlphaScreen SureFire ERK Assay
    • Sample Protocol
    • Assay Formats
    • Assay Optimization
    • Helpful Hints for Performing SureFire AlphaScreen ERK Assays
    • Websites
    • References
  • IP-3/IP-1 Assays
    Kim E. Garbison, Beverly A. Heinz, and Mary E. Lajiness.
    Published May 1, 2012.
    • Abstract
    • Introduction
    • Overview of Technology
    • IP-One HTRF^® Technology (Cisbio)
    • Sample Preparation Protocol
    • Results and Data Analysis
    • Assay Formats
    • Assay Optimization
    • Web Sites
    • References
  • Cardiomyocyte Impedance Assays
    Sarah D. Lamore, Clay W Scott, and Matthew F. Peters.
    Published February 25, 2015.
    • Abstract
    • Flowchart
    • Introduction
    • Concept and Overview of xCELLigence RTCA Cardio System
    • Sample Protocol
    • Assay Optimization
    • References
  • Screening for Target Engagement using the Cellular Thermal Shift Assay - CETSA
    Hanna Axelsson, Helena Almqvist, Brinton Seashore-Ludlow, and Thomas Lundbäck.
    Published July 1, 2016.
    • Abstract
    • Flow Chart
    • Introduction
    • Assay Design
    • Assay Development
    • Assay Validation
    • Materials and Reagents
    • Reduction of Assay Variability and Troubleshooting
    • Tentative Applications
    • References
  • Measurement of cAMP for G_αs- and G_αi Protein-Coupled Receptors (GPCRs)
    Tao Wang, Zhuyin Li, Mary Ellen Cvijic, Litao Zhang, and Chi Shing Sum.
    Published November 20, 2017.
    • Introduction
    • Flowchart of Assay Development Guidelines
    • Overview of the Assay Technology
    • Importance of cAMP Standard Curve
    • Assay Development and Optimization Guidelines
    • cAMP Assays for Receptors Coupled to G_αs Protein
    • cAMP Assays for Receptors Coupled to G_αi Protein
    • Acknowledgements
    • References
  • Measurement of β-Arrestin Recruitment for GPCR Targets
    Tao Wang, Zhuyin Li, Mary Ellen Cvijic, Carol Krause, Litao Zhang, and Chi Shing Sum.
    Published November 20, 2017.
    • Introduction
    • Flowchart of Assay Development Guidelines
    • Overview of the β-Arrestin Recruitment Assay
    • Guidelines and Sample Protocol for Agonist Assays
    • Guidelines and Sample Protocol for Antagonist Assays
    • Troubleshooting
    • Acknowledgements
    • References
  • Genome Editing Using Engineered Nucleases and Their Use in Genomic Screening
    Joana R. Costa, Bruce E. Bejcek, James E. McGee, Adam I. Fogel, Kyle R. Brimacombe, and Robin Ketteler.
    Published November 20, 2017.
    • Abstract
    • Introduction
    • Screening
    • Pooled Library Screening
    • Arrayed Library Screening
    • Conclusion
    • Appendix
    • References
    • Suggested Reading
    • Suggested Websites
  • Inhibition of Protein-Protein Interactions: Cell-Based Assays
    Mark Wade, Jacqui Méndez, Nathan P. Coussens, Michelle R. Arkin, and Marcie A. Glicksman.
    Published November 20, 2017.
    • Abstract
    • Introduction
    • Background
    • General Considerations for Developing Cell-Based PPI Assays
    • Förster Resonance Energy Transfer (FRET)
    • Bioluminescence Resonance Energy Transfer (BRET)
    • Fluorescent Protein Fragment Complementation
    • Luciferase Enzyme Fragment Complementation
    • Alternative Enzyme Complementation Assays
    • Suggested Websites
    • References
  • Pharmacological Characterization of GPCR Agonists, Antagonists, Allosteric Modulators and Biased Ligands from HTS Hits to Lead Optimization
    Chi Shing Sum, Brian J. Murphy, Zhuyin Li, Tao Wang, Litao Zhang, and Mary Ellen Cvijic.
    Published November 1, 2019.
    • Abstract
    • Introduction to GPCRs as Drug Targets
    • GPCR Signaling from a Drug Discovery Perspective
    • High-Throughput Screening Assays for Identification and Optimization of GPCR Ligands
    • Pharmacological Deliverables of HTS and Lead Optimization
    • Important Pharmacological Considerations During Lead Optimization
    • How the On- and Off-Rates of the Test Agonist (Compound Kinetics) Affect Compound Functional Activity Measurements and Thereby Impact Lead Optimization
    • Dose-Shift Assays
    • Dose-Shift Assays in Screening Mode
    • Biased Signaling
    • Conclusion
    • Acknowledgement
    • Keywords
    • References
• In Vivo Assay Guidelines
  Editors: Viswanath Devanarayan and Philip W. Iversen.
  • 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.
    • Flow Chart
    • 1. Introduction
    • 2. Assay Validation Procedures
    • 3. Design, Sample Size, Randomization, and Analysis Considerations
    • 4. Abbreviations
    • 5. Suggested Reading
  • In Vivo Receptor Occupancy in Rodents by LC-MS/MS
    Cynthia D. Jesudason, Susan DuBois, Megan Johnson, Vanessa N. Barth, and Anne B. Need.
    Published March 9, 2017.
    • Abstract
    • Experimental Design
    • Flowchart
    • Materials and Methods
    • References
• Assay Artifacts and Interferences
  Editors: Nathan P. Coussens and Jayme L. Dahlin.
  • Compound-Mediated Assay Interferences in Homogeneous Proximity Assays
    Nathan P. Coussens, Douglas Auld, Philippe Roby, Jarrod Walsh, Jonathan B. Baell, Stephen Kales, Kamyar Hadian, and Jayme L. Dahlin.
    Published February 1, 2020.
    • Abstract
    • Flowchart
    • Abbreviations
    • Introduction to Proximity Assays
    • Chemical Mechanisms of Assay Interference in Homogeneous Proximity Assays
    • Strategies to Identify Compound-Mediated Interference in Homogeneous Proximity Assays
    • Strategies to Mitigate Compound-Mediated Interferences in Proximity Assays
    • Conclusions
    • Suggested Readings (alphabetical order)
    • Suggested Web Resources
    • Glossary
    • Acknowledgements
    • Conflict of Interest Statement
    • References
  • Assay Interference by Chemical Reactivity
    Jayme L. Dahlin, Jonathan Baell, and Michael A. Walters.
    Published September 18, 2015.
    • Abstract
    • Flow Chart
    • Abbreviations
    • Introduction and Background
    • Knowledge-Based Strategies to Minimize Impact of Interference Compounds
    • Experimental-Based Strategies to Mitigate the Impact of Reactive Interference Compounds
    • Conclusions
    • Suggested Websites
    • Suggested Readings (alphabetical order)
    • References
  • Interference with Fluorescence and Absorbance
    Anton Simeonov and Mindy I. Davis.
    Published December 7, 2015; Last Update: July 1, 2018.
    • Abstract
    • Flow Chart
    • Fluorescence Interferences
    • Absorbance Interferences
    • Acknowledgements
    • References
  • Interferences with Luciferase Reporter Enzymes
    Douglas S. Auld and James Inglese.
    Published July 1, 2016; Last Update: July 1, 2018.
    • Abstract
    • Introduction
    • Interferences with Reporter Enzymes
    • Strategies to Mitigate Luciferase Inhibitor Interference
    • References
  • Assay Interference by Aggregation
    Douglas S. Auld, James Inglese, and Jayme L. Dahlin.
    Published July 26, 2017.
    • Abstract
    • Flowchart
    • Abbreviations
    • Introduction and Background
    • Strategies to Mitigate Incidence of Aggregation in Biochemical Assays
    • Strategies to Identify Aggregation
    • Frequently Asked Questions
    • Conclusions
    • Suggested Web Resources
    • Suggested Readings (alphabetical order)
    • Acknowledgements
    • REFERENCES
• Assay Validation, Operations and Quality Control
  Editors: Viswanath Devanarayan, Philip W. Iversen, Jeffrey R. Weidner, and Mary Jo Wildey.
  • Basic Guidelines for Reporting Non-Clinical Data
    Jayme L. Dahlin, G. Sitta Sittampalam, Nathan P. Coussens, Viswanath Devanarayan, Jeffrey R. Weidner, Philip W. Iversen, Joseph V. Haas, Duane D. Bronson, O. Joseph Trask, Jr., Todd R. Wiernicki, and Steven D. Kahl.
    Published October 1, 2019.
    • Abstract
    • Abbreviations
    • Overview
    • Rounding to Decimal Places
    • Rounding to Significant Digits
    • Presenting Rounded Data in Tables
    • Logarithms
    • Statistical Descriptors/Metrics
    • Example of Methods to Plot the Same Data
    • Correlation of Data
    • Agreement Between Two Variables
    • P-values
    • Calculation of Upper and Lower Quantitation Limits
    • Area under the Curve
    • Graph Elements
    • Types of Graphs
    • Graphing/Statistical Software Programs
    • Tables
    • Interactive Data Visualization
    • Databases
    • References
  • 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.
    • Abstract
    • Introduction
    • The BAO to organize screening assays, results and campaigns
    • Ontology development and overview of the BAO
    • Application of the BAO
    • Assay Annotation Tool to facilitate adaptation of the BAO
    • BAOSearch software application
    • Summary and conclusion
    • Acknowledgements
    • References
    • Glossary of BAO terms (partial list)
  • 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 20, 2017.
    • Abstract
    • Introduction
    • Data Types and Associated Rules for Radioligand Binding Assays: Inhibition Mode
    • Data Types and Associated Rules for Enzymatic Assays: Inhibition Mode
    • Data Types and Associated Rules for In Vitro Functional Assays
    • Reference
    • Glossary
  • 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.
    • Abstract
    • 1. Overview
    • 2. Stability and Process Studies
    • 3. Plate Uniformity and Signal Variability Assessment
    • 4. Replicate-Experiment Study
    • 5. How to Deal with High Assay Variability
    • 6. Bridging Studies for Assay Upgrades and Minor Changes
    • 7. References
  • 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: November 20, 2017.
    • Abstract
    • A. Determination of EC₅₀/IC₅₀
    • B. Production Monitoring
    • References
  • Minimum Significant Ratio – A Statistic to Assess Assay Variability
    Joseph V. Haas, Brian J. Eastwood, Philip W. Iversen, Viswanath Devanarayan, and Jeffrey R. Weidner.
    Published November 1, 2013; Last Update: November 20, 2017.
    • Abstract
    • 1. Introduction
    • 2. Common Types of MSR
    • 3. Other Considerations
    • 4. Conclusion
    • 5. References
• Assay Technologies
  Editors: Marcie Glicksman, James McGee, Owen McManus, and G. Sitta Sittampalam.
  • HPLC-MS/MS for Hit Generation
    Stefan Jon Thibodeaux, David A Yurek, and James E McGee.
    Last Update: July 8, 2017.
    • Abstract
    • Introduction
    • Chromatography
    • Instrument Set Up
    • Data Handling
    • Other Notes
    • References
  • Impedance-Based Technologies
    Kim E. Garbison, Beverly A. Heinz, Mary E. Lajiness, Jeffrey R. Weidner, and G. Sitta Sittampalam.
    Published May 1, 2012.
    • Abstract
    • Introduction
    • Overview of ACEA Biosciences RT-CES^®
    • Overview of CellKey^TM System
    • Sample Protocol for CellKey^TM (MDS Analytical Technologies)
    • CellKey^TM System Results and Data Analysis
    • Helpful Hints for Performing CellKey^TM Assays
    • Additional References
    • Acknowledgements
• Instrumentation
  Editors: Nathan P. Coussens, Sarine Markossian, and G. Sitta Sittampalam.
  • Microplate Selection and Recommended Practices in High-throughput Screening and Quantitative Biology
    Douglas S. Auld, Ph.D., Peter A. Coassin, B.S., Nathan P. Coussens, Ph.D., Paul Hensley, Carleen Klumpp-Thomas, Sam Michael, G. Sitta Sittampalam, Ph.D., O. Joseph Trask, B.S., Bridget K. Wagner, Ph.D., Jeffrey R. Weidner, Ph.D., Mary Jo Wildey, Ph.D., and Jayme L. Dahlin, M.D., Ph.D.
    Published June 1, 2020.
    • Abstract
    • Key Abbreviations
    • Introduction to Microplates in High-Throughput Screening and Quantitative Biology
    • Technical Factors in Microplate Selection
    • Technology-Specific Considerations – High-Content Screening
    • Microplate Usage
    • Troubleshooting and Special Topics
    • Conclusions
    • Suggested Readings (alphabetical order)
    • Glossary
    • Acknowledgements
    • Conflict of Interest Statement
    • References
  • Basics of Assay Equipment and Instrumentation for High Throughput Screening
    Eric Jones, Sam Michael, and G. Sitta Sittampalam.
    Published May 1, 2012; Last Update: April 2, 2016.
    • Abstract
    • Introduction
    • Common Equipment in HTS Labs
    • Microplates
    • Microplate Sealing
    • Microplate Readers
    • Spectrophotometry
    • pH Meters
    • Electronic Balances
    • Microscopes
    • Liquid Handling Devices
    • Suggested Websites and Resources
    • Suggested Readings (alphabetical order)
  • 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.
    • Abstract
    • Introduction
    • Radioactive Calculations
    • Instrumentation
    • Uniformity Plate
    • Color Quench Correction
    • Abbreviations
• Pharmacokinetics and Drug Metabolism
  Editors: Thomas D.Y. Chung and Xin Xu.
  • In Vitro and In Vivo Assessment of ADME and PK Properties During Lead Selection and Lead Optimization – Guidelines, Benchmarks and Rules of Thumb
    Thomas D.Y. Chung, David B. Terry, and Layton H. Smith.
    Published September 9, 2015.
    • Abstract
    • Flow Chart of a Two-tier Approach for In Vitro and In Vivo Analysis
    • Background
    • In Vitro Analysis - Low Compound Requirements and Relative Moderate Capacity
    • Example of In Vitro ADME Profiling Assays
    • In Vivo Analysis - High Compound Requirements and Low Capacity
    • Suggested Equipment and Resources
    • References
• Glossary
  Editors: Nathan P. Coussens, Sarine Markossian, G. Sitta Sittampalam, and Jeffrey R. Weidner.
  • 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 April 15, 2012; Last Update: September 22, 2014.
    • Abstract
    • Cell Culture Terms
    • Quantitative Biology Terms
    • Genetics Terms

Previous Editors: Bruce Bejcek, Jose M.M. Caaveiro, Neely Gal-Edd, Steven D. Kahl, Stephen C. Kales, Susan Kirshner, Vance P. Lemmon, Lisa Minor, Andrew Napper, Henrike Nelsen, John M. Peltier, Yueming Wang

Contact Information: vog.hin@naissokram.eniras