Table of Contents
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 1
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
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
14.6 Current Stage of Microfluidic Technology in Bioassays 411
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
