Contents:

Section Summary 1 Executive Summary 2 Introduction 2.1 Definitions 2.1.1 Assays and Analytes 2.1.2 Qualitative and Quantitative 2.1.3 Targets 2.1.4 Stains, Dyes, Tags, Labels, and Reporters 2.1.5 Potentially Confusing Terminology 2.2 Brief Historical Perspective 2.2.1 Diagnostics 2.2.2 Drug Discovery 2.3 Basic Issues 2.3.1 Sampling 2.3. 2 Primary and Secondary Assays 2.3.3 Confounding Analytes 2.4 Drug Development and Manufacturing 2.4.1 Impurities 2.4.2 Therapeutics 2.5 Miniaturization 2.5.1 Microplates 2.5.2 Arrays and Microarrays 3 Common Assays 3.1 Quantitation of Therapeutics and Other Compounds 3.1.1 Weighing 3.1.2 Extinction Coefficient 3.1.3 Evaporative Light Scattering Detection 3.1.4 Nuclear Magnetic Resonance 3.1.5 Chemiluminescent Nitrogen Detection 3.2 Microbial Contamination 3.2.1 Culture Tests 3.2.2 PCR 3.2.3 ELISA 3.3 Binding 3.3.1 Molecular Size 3.3.2 Biochemical Function 3.3.3 Labeling 3.4 Enzyme Assays 3.4.1 Proteases 3.4.2 Kinases and Phosphorylases 3.5 G-Protein Coupled Receptors 3.5.1 Cell Culture Expression 3.5.2 Promiscuous GPCR 3.5.3 cAMP 3.5.4 Calcium and IP3 3.6 Ion Channels 3.6.1 Ion Flux 3.6.2 Patch-Clamp 3.6.3 Reporter Dyes 3.6.4 Voltage Sensitive Dye Systems 3.6.5 Membrane Binding Assays 3.7 Toxicology and Pharmacology 3.7.1 Ion Channel Assays in Cardiac Toxicity 3.7.2 Gene Expression in ADME 3.7.3 Hepatotoxicity 3.8 Genetic Polymorphism 3.8.1 Restriction Fragment Length Polymorphisms 3.8.2 Hybridization Assays 3.8.3 PCR for SNP 3.8.4 Single Base Extension 4 General Assay Design 4.1 Universal Considerations 4.1.1 Precision 1 4.1.2 Optimal Reagent Amounts 4.1.3 Standard Curves 4.1.4 Multiplicity and Statistics 4.1.5 Interpolation 4.2 Nature of Analyte and Matrix 4.2.1 Analyte Stability 4.2.2 Separation and Enrichment 4.2.3 Internal Standard "Spike" 4.3 Assay Objectives 4.3.1 Research, Development, or Process 4.3.2 Budget 4.3.3 Throughput 4.3.4 Scalability 4.3.5 Sensitivity 4.3.6 Error Tolerance 5 Format 5.1 Homogenous and Heterogeneous 5.1.1 Pros and Cons 5.1.2 Heterogeneous Immobilization 5.2 Direct and Indirect 5.2.1 Second Mediators 5.2.2 Second Antibodies 5.2.3 Biotin-Avidin 5.2.4 Enzyme Reporter Systems 5.3 Agonists and Antagonists 5.3.1 Competition 5.4 In Vitro and In Vivo 5.4.1 In Vitro Assays 5.4.2 In Vivo Assays 5.4.3 Biological Material 6 Readout (Reporting Format) 6.1 Colorimetric and Fluorometric 6.1.1 Colorimetric Assays 6.1.2 Fluorometric Assays 6.2 Radiometric 6.2.1 Radiolabeling 6.2.2 Radioimmunoassay 6.2.3 Scintillation Proximity Assay 6.3 Biological Growth 6.3.1 Special Growth Media 6.4 Other Readouts 7 Validation 7.1 Installation and Operation Qualification 7.1.1 Instruments and Equipment 7.1.2 Assay Optimization 7.1.3 Other Variables 7.2 Performance Qualification 7.2.1 Instruments and Consumables 7.2.2 Operation Re-Certification 7.2.3 Assay Parameters 7.2.4 Data Certification 7.3 Scale-Up 7.3.1 General 7.3.2 Step-by-Step Process 8 In Vitro Assays 8.1 General 8.1.1 Spectroscopy 8.1.2 Protein Assays 8.2 Enzyme Assays 8.2.1 Enzyme Stability 8.2.2 Proteases 8.2.3 Kinases and Phosphorylases 8.2.4 Enzyme SPA 8.3 Binding 8.3.1 Specificity 8.3.2 Valence and Avidity 8.3.3 Blocking and Washing 8.4 Antibodies and Immunoassays 8.4.1 Antibodies 8.4.2 Immunoassays 8.4.3 ELISAs 8.4.4 "Classical" Immunoassays 8.4.5 Fluorescence Quenching 8.4.6 Fluorescence Polarization 8.4.7 Scintillation Proximity Assay 8.4.8 Other Binding Assays 8.5 Nucleic Acids 8.5.1 General 8.5.2 Polymerase Chain Reaction 8.5.3 Hybridization Blots 9 Cell-Based Assays 9.1 General 9.1.1 Pitfalls 9.1.2 Controls 9.2 Fixed Cells 9.2.1 Fixing 9.2.2 Fluorescence in Situ Hybridization 9.2.3 Immunofluorescence Assay 9.2.4 Staining and Counter-Staining 9.3 Whole Cells 9.3.1 Cell ELISAs and ELISpots 9.3.2 Cytotoxicity 9.3.3 Surface Binding and Membrane Transport 9.3.4 Trafficking and Translocation 9.3.5 Nuclear Receptors 9.3.6 Cell Quantitation 9.3.7 Vitality 9.3.8 Motility 9.3.9 Apoptosis 9.3.10 SPA in Microplates 9.3.11 Fluorescence 9.4 Flow Cytometry 9.4.1 General 9.4.2 Advantages and Limitations 9.4.3 Fluorophore Selection 9.4.4 Controls 9.4.5 Gating 9.4.6 Applications 9.5 Lysates 9.5.1 Gene Expression 9.5.2 Protein Expression (Gene Induction) 9.6 Microscopy 9.6.1 Stains 9.6.2 Labels 9.6.3 FISH and IFA 10 Automation Platforms 10.1 Miniaturization 10.1.1 Pros and Cons 10.1.2 Scaling 10.2 Robotics 10.2.1 Conveyors and Workstations 10.2.2 Integrated and Modular 10.2.3 Examples 10.3 Liquid Handlers 10.3.1 General Applications 10.3.2 Delivery Size 10.3.3 Calibration 10.3.4 Cross-Contamination 10.3.5 Pipet Tips 10.4 Cell Handling 10.4.1 Culture 10.4.2 Sample Prep 10.5 Microplate Equipment 10.5.1 Microplates 10.5.2 Plate Handlers 10.5.3 Washers 10.5.4 Bar Codes 10.5.5 Cleaning and Maintenance 11 Emerging Technologies 11.1 Automated Image Analysis 11.1.1 General Issues 11.1.2 Practical Considerations 11.1.3 An Example 11.2 Higher Density Formats 11.2.1 384-Well Plates 11.2.2 High Density Microplates 11.2.3 Microarrays, Microfluidics, and Chips 11.3 New Technologies 11.3.1 Surface Plasmon Resonance 11.3.2 Flow Cytometry with Labeled Beads and Libraries 11.3.3 Branched DNA Binding Assay 11.3.4 Single Molecule Detection 11.3.5 Virtual Screening 12 Information Management 12.1 Data Analysis 12.1.1 Acquisition 12.1.2 Signal-To-Background and Signal-To-Noise Ratios 12.1.3 Precision and Accuracy 12.1.4 Type 1 and Type 2 Error 12.1.5 Random and Systematic Error 12.1.6 Binning and Pooling 12.2 Statistics 12.2.1 Binding Constants 12.2.2 Michaelis-Menten Equations 12.2.3 Error and Standard Error 12.2.4 Correlation Coefficient 12.2.5 Standard Deviation 12.2.6 Coefficient of Variance 12.2.7 Z' Factor 12.2.8 More Complicated Statistics 13 Appendices 13.1 Resources for Detailed Protocols 13.1.1 Orgs 13.1.2 Journals and Other Commercial Publications 13.2 Checklist for General Assay Development. 13.3 Microarray Assay Checklist 13.4 Vendors 13.5 Examples of Troubleshooting 13.5.1 In Vitro 13.5.2 In Vivo 14 Bibliography