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Assay Guidance Manual
Introduction
Assay Validation
Assay Operations for SAR Support
Enzymatic Assays
Receptor Binding Assays
GTPγS Binding Assays
Tissue Culture Assays
Cell-Based Elisa (C-Elisa) and Westerns Blots for Quantitative Antigen Detection
FLIPR™ Assays to Measure GPCR and Ion Channel Targets
Immunoassay Methods
Data Standardization for Results Management
Mechanism of Action Assays for Enzymes
Glossary of Quantitative Biology Terms
NCGC Assay Guidance Criteria
Table of Contents
Copyright © 2008, Eli Lilly and Company and the National Institutes of Health Chemical Genomics Center. All Rights Reserved. For more information, please review the Privacy Policy and Site Usage and Agreement.

How to cite this document:
Assay Guidance Manual Version 5.0, 2008, Eli Lilly and Company and NIH Chemical Genomics Center.
Available online at: http://www.ncgc.nih.gov/guidance/manual_toc.html (last accessed [insert date here])

SECTION I: INTRODUCTION
  1. INTRODUCTION
SECTION II: TRANSFER OF VALIDATED ASSAYS
  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
SECTION III: ASSAY OPERATIONS FOR SAR SUPPORT
  1. DETERMINATION OF EC50/IC50
  2. PRODUCTION MONITORING
SECTION IV: ENZYMATIC ASSAYS
  1. ENZYME ASSAY DEVELOPMENT FLOW CHART
  2. INTRODUCTION
  3. CONCEPT
  4. REAGENTS AND METHOD DEVELOPMENT
  5. DETECTION SYSTEM LINEARITY
  6. ENZYME REACTION PROGRESS CURVE
  7. MEASUREMENT OF KM AND VMAX
  8. DETERMINATION OF IC50 FOR INHIBITORS
  9. INHIBITION CONSTANT (KI)
  10. IC50 DETERMINATION FOR SAR
  11. OPTIMIZATION EXPERIMENTS
  12. ASSAY VALIDATION
SECTION V: RECEPTOR BINDING ASSAYS
  1. STEPS TO ASSAY DEVELOPMENT FOR SPA FORMAT
  2. STEPS TO ASSAY DEVELOPMENT FOR FILTER FORMAT
  3. INTRODUCTION
  4. REAGENTS
  5. SCINTILLATION PROXIMITY ASSAYS (SPA)
  6. SPA ASSAY FORMAT
  7. ASSAY BUFFER
  8. ASSAY CONDITIONS
  9. BINDING PARAMETERS
  10. FILTRATION ASSAYS
  11. FILTER ASSAY FORMAT
  12. ASSAY BUFFER
  13. ASSAY CONDITIONS
  14. BINDING PARAMETERS
  15. SPECIAL CIRCUMSTANCES
  16. PRACTICAL USE OF FLUORESCENCE POLARIZATION IN COMPETITIVE RECEPTOR BINDING ASSAYS
  17. ABBREVIATIONS
  18. ADDITIONAL RESOURCES
SECTION VI: GTPγS BINDING ASSAYS
  1. STEPS TO ASSAY DEVELOPMENT
  2. INTRODUCTION
  3. MATERIALS AND REAGENTS
  4. MEMBRANE PREPARATIONS AND ASSAY BUFFERS
  5. BASIC ASSAY PROTOCOL
  6. ASSAY OPTIMIZATION
  7. DATA ANALYSIS
  8. FILTRATION ASSAYS
  9. NON-RADIOMETRIC GTPγS ASSAYS
  10. LITERATURE REFERENCES
SECTION VII: TISSUE CULTURE ASSAYS
  1. CELL CULTURE GLOSSARY
  2. INTRODUCTION TO CELL BASED ASSAYS
  3. REQUIREMENTS FOR ASSAY PROOF OF CONCEPT FOR CELL BASED ASSAYS
  4. TISSUE CULTURE ASSAY DEVELOPMENT, OPTIMIZATION AND VALIDATION FLOW CHART
  5. TYPES OF TISSUE CULTURE ASSAYS
  6. BATCH CO-TRANSFECTION AND CELL BANKING PROTOCOLS
  7. CRYOPRESERVATION OF CELLS
  8. CELL COUNTING PROTOCOLS
  9. FACTORS FOR CELL BASED ASSAY DEVELOPMENT
  10. TYPICAL CELL CULTURE PROTOCOL
  11. STANDARD HTS CELL CULTURE PRACTICES TO REDUCE CONTAMINATION RISK
  12. EXAMPLE, TRANSIENT TRANSFECTION OF CELLS
  13. TRANSIENT TRANSFECTION METHODS
  14. TRANSFECTION CATIONIC MATRIX REAGENTS
  15. GENERAL CELL BIOLOGY INFORMATION
  16. REFERENCES
SECTION VIII: CELL-BASED ELISA (C-ELISA) AND WESTERNS BLOTS FOR QUANTITATIVE ANTIGEN DETECTION
  1. INTRODUCTION
  2. CELL CULTURE AND TREATMENTS
  3. FIXING AND STAINING
  4. REAGENTS
  5. ASSAY VALIDATION
  6. APPENDIX 1. IMMUNOBLOT VS. CYTOBLOT ASSAYS FOR ENZYME INHIBITOR POTENCIES
  7. APPENDIX 2. LINEARITY AND SENSITIVITY OF CELL-BASED WESTERN ASSAYS
SECTION IX: FLIPR™ ASSAYS TO MEASURE GPCR AND ION CHANNEL TARGETS
  1. OVERVIEW: FLIPR™ ASSAY DEVELOPMENT
  2. INTRODUCTION
  3. TYPES OF FLIPR™ FORMATS
  4. REAGENTS AND BUFFERS FOR METHOD DEVELOPMENT
  5. METHOD DEVELOPMENT AND OPTIMIZATION
  6. FLIPR INSTRUMENT SET-UP
  7. POTENTIAL ARTIFACTS
  8. REFERENCES
SECTION X: IMMUNOASSAY METHODS
  1. IMMUNOASSAY DEVELOPMENT, OPTIMIZATION AND VALIDATION FLOW CHART
  2. INTRODUCTION
  3. IMMUNOASSAY PARAMETERS
  4. REAGENTS
  5. INSTRUMENTATION
  6. IMMUNOASSAY FORMATS
  7. IMPORTANT PARAMETERS FOR DEVELOPMENT OF AN IMMUNOASSAY
  8. INITIAL CONCEPT AND METHOD DEVELOPMENT OF A SANDWICH IMMUNOASSAY
  9. RESULTS
  10. INITIAL CONCEPT AND METHOD DEVELOPMENT OF A COMPETITIVE ASSAY
  11. DEVELOPMENT OF A COMPETITIVE IMMUNOASSAY
  12. METHOD VALIDATION (PRE-STUDY)
  13. METHOD VALIDATION (IN-STUDY)
  14. PRE-STUDY & IN-STUDY ACCEPTANCE CRITERIA
  15. REFERENCES
SECTION XI: DATA STANDARDIZATION FOR RESULTS MANAGEMENT
  1. INTRODUCTION
  2. DATA TYPES AND ASSOCIATED RULES FOR RADIOLIGAND BINDING ASSAYS: INHIBITION MODE
  3. ATA TYPES AND ASSOCIATED RULES FOR ENZYMATIC ASSAYS: INHIBITION MODE
  4. DATA TYPES AND ASSOCIATED RULES FOR IN VITRO FUNCTIONAL ASSAYS
  5. GLOSSARY
SECTION XII: MECHANISM OF ACTION ASSAYS FOR ENZYMES
  1. GLOSSARY OF MOA TERMS
  2. OVERVIEW OF MOA IN DRUG DISCOVERY
  3. TYPES OF INHIBITION
  4. PERFORMING MOA STUDIES
  5. REFERENCES
SECTION XIII: GLOSSARY OF QUANTITATIVE BIOLOGY TERMS
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