Assay development : fundamentals and practices

FOREWORD xi PREFACE xiii ACKNOWLEDGMENTS xv ABBREVIATIONS xvii 1 INTRODUCTION TO ASSAY DEVELOPMENT 1 1.1 Assay and Bioassay 1 1.2 Drug Discovery Process and Role of Assays in the Process 7 1.3 Bioassay Development 17 1.4 Bioassay Classifications 22 Useful Websites 24 Bibliography 24 2 MEASUREMENT AND INSTRUMENTATION 27 2.1 Measurement and Perturbation 27 2.2 Common Instrumental Methods and Instrument Components 32 2.3 Molecular Absorption Measurements 39 2.4 Molecular Luminescence Measurements 43 2.5 Luminescence Lifetime Measurement and Time-Resolved Fluorescence Measurement 53 2.6 Fluorescence Resonance Energy Transfer (FRET) and Time-Resolved-FRET 54 2.7 Fluorescence Quenching 57 2.8 Fluorescence Polarization (FP) 58 2.9 Radioactivity Measurement 61 2.10 Evaluating and Selecting an Instrumental Method for Bioassay 64 Useful Websites 68 Bibliography 68 3 FUNDAMENTAL PRINCIPLES OF ASSAY DEVELOPMENT WITH ISOLATED PROTEINS 69 3.1 Chemical Potential, Equilibrium, and Kinetics 70 3.2 Protein Binding Studies at Equilibrium 72 3.3 Kinetic Studies of the Protein Binding Process 77 v 3.4 Enzyme Kinetics 83 3.5 Inhibition of Protein Function 89 3.6 Assay Development with Isolated Proteins 98 Useful Websites 103 Bibliography 104 4 SEPARATION-BASED TECHNIQUES IN BIOASSAYS 105 4.1 Washing Solid Supports to Remove Impurities 105 4.2 Organic Solvent Extraction of Hydrophobic Molecules 107 4.3 Centrifugation to Remove Dense Particles 108 4.4 Membrane Filtration 109 4.5 Liquid Chromatography 110 4.6 Electrophoresis 117 Useful Websites 122 Bibliography 122 5 GENERAL PROTEIN BINDING ASSAY FORMATS 125 5.1 Equilibrium Dialysis 126 5.2 Competitive Binding Assays with Radioactive or Other Labeled Ligands 128 5.3 Application of SPA and FlashPlate in Binding Studies 131 5.4 Application of Fluorescence Polarization (FP) in Binding Studies 132 5.5 Application of FRET Assays in Binding Studies 137 5.6 Application of ELISA in Binding Studies 139 5.7 Surface Plasmon Resonance (SPR) Technology and Its Application in Binding Studies 143 5.8 Application of Label-Free Technologies in Binding Studies 151 Useful Websites 152 Bibliography 153 6 FUNCTIONAL ASSAYS WITH ISOLATED PROTEASES 155 6.1 Introduction to Proteases and Their Substrates 155 6.2 Function of Proteases and Their Role in Drug Discovery 159 6.3 Protease Assays 161 6.4 Protease Substrate Profiling 166 6.5 Protease Inhibitors 168 6.6 Assay Development for Caspases with a Fluorogenic Substrate 170 6.7 Assay Development for Carboxypeptidase U (EC 3.4.17.20) 173 Useful Websites for Proteases 176 Bibliography 177 7 FUNCTIONAL ASSAYS FOR PROTEIN KINASES 181 7.1 Introduction to Protein Kinases 181 7.2 Substrates for In Vitro Kinase Assays 182 7.3 Kinase Assay Development Strategies 186 7.4 Kinase Assay Based on Detection of Phosphorylated Product 187 7.5 Kinase Assays by Measuring the Generation of ADP 199 7.6 Kinase Assays by Measuring the Depletion of ATP 200 7.7 Kinase Assays by Measuring the Depletion of Peptide Substrate 204 7.8 Kinase Assays by Simultaneous Measurement of Both Product and Substrate 204 7.9 Example of a Kinase Assay Development in HTRF Format 205 Useful Websites 209 Bibliography 209 8 FUNDAMENTAL PRINCIPLES OF CELL-BASED ASSAYS 213 8.1 Cell Signaling, Signal Transduction, and Cellular Responses 214 8.2 General Approaches in Cell-Based Assays 221 8.3 Concept of Affinity and Efficacy in Cell-Based Assays 223 8.4 Development of Cell-Based Assays 228 Useful Websites 235 Bibliography 235 9 FUNCTIONAL ION CHANNEL ASSAYS 239 9.1 Introduction to Ion Channels 239 9.2 Strategies for Ion Channel Assays 243 9.3 Electrophysiological Methods 247 9.4 Ion Flux Methods 253 9.5 Membrane Potential Sensing Methods 259 9.6 Selecting Suitable Assays for Ion Channel Studies 261 Useful Websites and Vendors 262 Bibliography 262 10 ASSAYS WITH GPCRs 265 10.1 Introduction to GPCRs and G Proteins 265 10.2 G Protein-Coupled Receptor Activation and Signal Transduction 267 10.3 Strategies of GPCR Assay Development 269 10.4 G Protein-Coupled Receptor Assays by Measuring the Extent of GTP Binding to Ga 270 10.5 G Protein-Coupled Receptor Assays Based on Measurement of cAMP 272 10.6 G Protein-Coupled Receptor Assays Based on Measurement of Intracellular Inositol Phospholipids 276 10.7 G Protein-Coupled Receptor Assays Based on Measurement of Intracellular Ca2+ 277 10.8 G Protein-Coupled Receptor Assays Based on Measurement of MAPK Activity 278 10.9 G Protein-Coupled Receptor Assays with Reporter Gene 279 10.10 G Protein-Coupled Receptor Assays by Monitoring Events Leading to GPCR Internalization 282 Useful Websites 285 Bibliography 285 11 ASSAYS BASED ON INTEGRATED CELL SYSTEM PROPERTIES 289 11.1 Cell Viability, Proliferation, and Cytotoxicity Assays 289 11.2 Measurement of Extracellular Indicators of Cellular Metabolism 296 11.3 Measurement of Cell’s Effect on Electrical Impedance 298 11.4 Measurement of Protein Secretion from Cells 301 11.5 Measurement of Discoloration of Melanophore Cells 302 11.6 Measurement of Cell Motility 303 Useful Websites 304 Bibliography 304 12 HIGH-CONTENT CELL-BASED ASSAY WITH OPTICAL IMAGING TECHNIQUES 307 12.1 Sample Preparation 308 12.2 Cellular Image Collection 312 12.3 Image Abstraction, Analysis, and Data Management 316 12.4 Applications of iCHCS 318 Useful Websites 319 Bibliography 320 13 HIGH-THROUGHPUT SCREENING 321 13.1 Introduction 321 13.2 Molecular or Cellular Targets and Assay Development 325 13.3 Compound Library Management 326 13.4 Hardware Module 332 13.5 Software Module 344 13.6 HTS Operation Management 347 13.7 Building an HTS Operation for Biopharmaceutical Discovery 355 13.8 Quality Control and Data Analysis in Primary Screening 359 Useful Websites 368 Bibliography 369 14 CASE STUDY: DEVELOPMENT OF A MICROFLUIDIC-BASED KINASE ASSAY PLATFORM 371 14.1 Background of Microfluidic Technology and Its Application in Bioassays 371 14.2 The Original Mobility Shift Kinase Assay Format 377 14.3 Realizing the Flaws in the Original Kinase Assay Format 384 14.4 Searching for Alternative Kinase Assay Methods 398 14.5 Development of the Off-Chip Kinase Assay Format 400 14.7 Appendix: Poster Presented at the 2002 Society for Biomolecular Screening Annual Meeting: Analysis of Mobility Shift Data Obtained from Labchip in Kinase Assay 413 Useful Websites 421 Bibliography 421 INDEX 423 14.6 Current Stage of Microfluidic Technology in Bioassays 411