Preface xiii
Part I Background to Coupled Bioluminescent Assays 1
1 Introduction 3
1.1 Introduction to Coupled Bioluminescent Assays, 3
1.2 Luminescent Technologies of the Life Sciences, 4
1.3 Varieties of Fluorometric Assays, 7
1.3.1 Time-Resolved Fluorescence, 8
1.3.2 Fluorescence Resonance Energy Transfer, 8
1.3.3 Fluorescence Polarization, 9
1.4 Chemiluminescence and Bioluminescence, 10
1.4.1 Chemiluminescence, 11
1.4.2 Bioluminescence, 13
1.5 Common Bioluminescence Systems, 14
1.5.1 Firefly Luciferase, 14
1.5.2 Renilla Luciferase, 16
1.5.3 Bacterial Luciferases, 18
1.5.4 Aequorin, 19
1.5.5 Other Luciferases and Photoproteins, 20
1.6 A Coupled Bioluminescent Reaction, 22
1.7 Summary, 23
2 Coupled Bioluminescent Reactions in Practice 24
2.1 Principles of Coupled Bioluminescent Reactions, 24
2.1.1 Requirements for Successful Coupled Bioluminescent Assays, 24
2.2 Instrumentation and Equipment for Coupled Bioluminescent Assays, 25
2.2.1 Instrument Testing, 27
2.2.2 Instrument Features, 28
2.2.3 Luminescent Microplates, 31
2.3 Coupled Bioluminescent Assay Procedures and Precautions, 32
2.3.1 Coupled Enzyme Assays, 32
2.3.2 Steady-State Kinetics, 36
2.3.3 Coupled Bioluminescent Reactions, 38
2.3.4 Recommended Precautions for Coupled Bioluminescent Reactions, 44
2.4 Data Handling for Coupled Bioluminescent Assays, 45
2.4.1 Analysis of Coupled Bioluminescent Assay Results by Time-Dependent Linear Regression, 46
2.4.2 Performing the Linear Regression, 49
2.4.3 Outliers, 50
2.4.4 Special Considerations Relating to High-Throughput Screening, 52
2.5 Comparison of Coupled Bioluminescent Assays with Other Methods, 53
2.5.1 Coupled Bioluminescent Assays Versus Spectrophotometric Assays, 53
2.5.2 Coupled Bioluminescent Assays Versus Fluorescent Assays, 54
2.5.3 Coupled Bioluminescent Assays Versus Chemiluminescent Assays, 55
Part II Biomedical Applications of Coupled Bioluminescence 57
3 Coupled Bioluminescent Cytotoxicity Assays 59
3.1 Introduction, 59
3.2 Membrane Integrity Assays, 60
3.2.1 Radioactive Isotope Methods, 60
3.2.2 Other Labeling Methods, 64
3.2.3 General Enzyme-Release Methods, 65
3.3 A Coupled Bioluminescent Assay for Enzyme Release: The G3PDH-Release Assay, 70
3.3.1 Development and Features, 70
3.3.2 Performance Characteristics, 75
3.4 Viability Assays, 82
3.4.1 ATP-Release Assay, 82
3.4.2 Assays of Vital Cellular Functions, 82
3.4.3 Simple Cell-Growth Assays, 83
3.4.4 Total Enzyme-Release Methods, 84
4 The Role of Coupled Bioluminescent Assays in Kinase Screening and Study 85
4.1 The Many Roles of Kinases in Biology, 85
4.2 Current Standard Kinase Assays, 86
4.2.1 Fluorometric Kinase Assays, 87
4.2.2 Radiolabeling Kinase Assays, 88
4.2.3 Fluorometric Assays Involving Detection of ADP, 89
4.3 Coupled Bioluminescent Kinase Assays, 90
4.3.1 A Historical Note: The Coupled Bioluminescent Creatine Kinase Assay, 90
4.3.2 ATP Depletion Kinase Assays, 90
4.3.3 Reverse CB Kinase Assays, 98
4.3.4 Kinases as Participants in Other CB Assays, 102
4.4 Conclusions, 102
5 Coupled Bioluminescent Phosphatase Assays 103
5.1 Introduction, 103
5.2 Phosphatases, 103
5.2.1 Catalytic Properties of Phosphatases, 104
5.2.2 Classification of Phosphatases, 104
5.3 Contemporary Phosphatase Assay Technologies, 105
5.3.1 Fluorometric Phosphatase Assays, 106
5.3.2 Colorimetric Phosphatase Assays, 117
5.3.3 Chemiluminescent Phosphatase Assays, 119
5.3.4 Radiolabeling Phosphatase Assays, 120
5.4 CB Phosphatase Assays, 122
5.4.1 Assays Using Phosphorylated Luciferin, 122
5.4.2 Assays Based on the GPL Reaction Series, 122
5.4.3 Challenges in the Development of the GPL-Coupled Bioluminescent System for Phosphate Detection, 127
5.4.4 Potential Drawbacks of GPL Phosphatase Assays, 129
5.5 Conclusions, 130
6 Acetylcholinesterase 131
6.1 Introduction, 131
6.2 Established AChE Assay Methods, 133
6.3 Recent Developments in AChE Assay Methods, 134
6.4 Evaluation of Current AChE High-Throughput Screening Methods, 135
6.5 Coupled Bioluminescent Assays of AChE Activity, 136
6.6 Comparison of Coupled Bioluminescent and Other Methods of Measuring AChE Activity, 138
7 Measurement of Nitric Oxide Synthase Activity by Coupled Bioluminescence 139
7.1 Introduction, 139
7.1.1 Nitric Oxide Synthases, 139
7.1.2 The Nitrate Ion in Medicine and NO Biology, 141
7.1.3 NO Biology, 141
7.1.4 Interrelationship Among Nitrate, Nitrite, and NO, 142
7.1.5 Medical Aspects of Nitrate Biology, 143
7.2 Current NOS Assays, 144
7.2.1 Measuring NOS Activity by Assays for Nitrite and Nitrate, 144
7.2.2 "Direct" Assays of NOS Activity, 148
7.2.3 Coupled Enzymatic Assays of NOS Activity, 148
7.3 Conclusions, 149
8 The Coupled Bioluminescent Pyrophosphorolysis Assay 150
8.1 Introduction, 150
8.2 Genetic Variation in Modern Medicine, 150
8.3 DNA Complementarity, 152
8.3.1 DNA Hybridization, 152
8.3.2 Primer Extension in SNP Analysis, 153
8.3.3 Primer Degradation in SNP Analysis, 154
8.4 The READIT ® Pyrophosphorolysis Assay, 156
8.4.1 READIT ® : Description and Technical Aspects, 156
8.4.2 Performing the READIT ® Assay, 157
8.4.3 Competitive Position of READIT ® , 158
9 Coupled Luminescent Assays of G-Protein-Coupled Receptors 160
9.1 Introduction to G-Protein-Coupled Receptors, 160
9.1.1 Structure and Function of GPCRs, 161
9.1.2 GPCRs in Medicine, 165
9.2 GPCR Assay Methods, 166
9.2.1 Arrestins as Markers of GPCR Activity, 166
9.2.2 Calcium Quantification in GPCR Assays, 169
9.2.3 Quantitative cAMP Assays of GPCR Activity, 175
9.2.4 GPCR Assays Involving Inositol Triphosphate Detection, 182
9.2.5 GPCR Assays Involving GTP, 183
9.2.6 GPCR Assays Involving Reporter Genes, 186
9.2.7 Novel GPCR Assays and Other Strategies, 189
9.3 Summary, 190
10 Coupled Bioluminescent Protease Assays 191
10.1 Introduction, 191
10.1.1 Proteases, 191
10.2 Protease Assays, 193
10.2.1 Chromogenic Protease Assays, 194
10.2.2 Fluorometric Protease Assays, 194
10.2.3 Exotic Fluorometric Protease Assays, 197
10.3 Coupled Bioluminescent Protease Assays, 198
10.3.1 Protease Assays Using Luciferin Derivatives, 198
10.3.2 Coupled Bioluminescent Protease Assays Employing Recombinant Luminescent Proteins, 198
10.4 Summary, 199
11 Coupled Luminescent Assays Involving Aequorin 201
11.1 Introduction to Aequorin, 201
11.1.1 Aequorin as the First Photoprotein, 201
11.1.2 The Aequorin Reaction, 202
11.2 Detection of Calcium in Practice, 203
11.2.1 Fluorometric Quantification of Calcium, 203
11.2.2 Coupled Bioluminescent Quantification of Calcium Using Aequorin, 207
11.2.3 Real-Time Imaging of Calcium Flux with Aequorin, 208
11.2.4 Other Uses of Aequorin in Coupled Bioluminescent Assays, 208
12 Coupled Bioluminescent Reporter Assays 209
12.1 Introduction to Reporter Assays, 209
12.1.1 Brief History and Development of Reporter Assays, 210
12.1.2 Other Non-Luminescent Reporter Proteins, 211
12.2 Luciferases as Reporters of Promoter Activities, 212
12.2.1 The Modern Luminescent Reporter Assay, 213
12.2.2 Multicolor Light Emission by Luciferases, 214
12.2.3 Dual-Mode Reporter Assays in Practice, 214
12.3 Aequorin as a Reporter Enzyme, 215
12.4 Vectors for Use in Reporter Assays, 216
12.4.1 Systems for Study of Specific Transcriptional Modulators, 216
12.4.2 Bacterial Reporter Systems, 216
12.4.3 Viral Vectors, 217
12.5 Summary, 217
13 Coupled Bioluminescent Assays: Regulatory Concerns 218
13.1 Introduction, 218
13.2 Regulatory Aspects of Assay Development, 220
13.2.1 Standard Operating Procedures, 220
13.2.2 Philosophy of Assay Validation, 221
13.2.3 Parameters for Assay Validation, 223
13.2.4 Gaining Regulatory Acceptance of CB Methods, 240
13.3 Summary, 240
Part III other Applications of Coupled Bioluminescence 241
14 Coupled Bioluminescent Determination of Bioburden and Sterility 243
14.1 Introduction, 243
14.2 Rapid Methods of Bioburden and Sterility Assessment, 244
14.2.1 Bioburden Measurement by ATP-Release Assay, 244
14.2.2 Bioburden Measurement by Protein Assay, 245
14.2.3 Coupled Bioluminescent Methods of Measuring Bioburden, 245
15 Environmental Applications of Coupled Bioluminescent Assays 247
15.1 Introduction, 247
15.2 Current Methods for Environmental Monitoring of Water Quality, 248
15.3 Methods of Monitoring Stream Water and Lake Water, 249
15.3.1 Monitoring of Phosphate in Freshwater, 249
15.3.2 Monitoring of Nitrate in Groundwater, 250
15.3.3 Monitoring of Pesticides in Freshwater, 251
15.3.4 Monitoring of Bacterial Content of Freshwater, 252
15.4 Methods of Monitoring Drinking Water, 254
15.4.1 Current Drinking Water Reports, 254
15.4.2 Biohazard Monitoring in Drinking Water, 255
15.4.3 Monitoring of Chemical Hazards in Drinking Water, 257
Appendix A: One-Letter Amino Acid Abbreviations 259
Glossary 260
Bibliography 265
Index 291
