Contents:

Chapter 1: Executive Summary Purpose and Scope Summary Additional Information Chapter 2: Introduction. Definitions Assays and Analytes Qualitative and Quantitative Targets Stains, Dyes, Tags, Labels, and Reporters Potentially Confusing Terminology Polarization Quenching Brief Historical Perspective Diagnostics Drug Discovery Basic Issues Sampling Primary and Secondary Assays High- throughput Versus High-content Different Error Tolerance Confounding Analytes Drug Development and Manufacturing Impurities Therapeutics Miniaturization Microplates 96-well Plates Evaporation Non-reproducibility Arrays and Microarrays Chapter 3: Common Assays Quantitation of Therapeutics and Other Compounds Weighing Extinction Coefficient. Evaporative Light Scattering Detection Nuclear Magnetic Resonance Chemiluminescent Nitrogen Detection Microbial Contamination Culture Tests Culture Media Validation and Archiving PCR ELISA Binding Molecular Size Surface Plasmon Resonance Biochemical Function Enzyme Reconstitution Labeling Enzyme Assays. Proteases Site-specific Proteases HPLC Protease Assays Fluorescence Based Protease Assays Color Based Protease Assays Gain of Biological Function. Kinases and Phosphorylases Radiometric Phosphate Assays Non-radiometric Phosphate Assays Chelation-based Phosphate Assays Phosphate Immunoassays G protein-coupled receptors Cell Culture Expression Promiscuous GPCR cAMP Calcium and IP3 Ion Channels Ion Flux Radiometry Atomic Absorption Spectroscopy Patch-clamp Reporter Dyes Voltage Sensitive Dye Systems Membrane Binding Assays Toxicology and Pharmacology Ion Channel Assays in Cardiac Toxicity Gene Expression in ADME Hepatotoxicity Genetic Polymorphism Restriction Fragment Length Polymorphisms Hybridization Assays PCR for SNP Single Base Extension Chapter 4: General Assay Design Universal Considerations Precision Laboratory Technique Reagent Purity Storage Blocking. Water Quality Cell and Tissue Quality Room Temperature Time Washing Other Sources of Non-reproducibility Repeatability between Labs Optimal Reagent Amounts Serial Dilutions Standard Curves Linearity Range Mathematical Modeling Limits Interpolation Data Analysis Multiplicity Signal-to-background and Signal-to-noise Ratios Z-prime Nature of Analyte and Matrix Analyte Stability Microbial Contamination Metabolic Processes Intractable Stability Problems Blocking Inadvertent Adsorption Separation and Enrichment Separation Enrichment Prederivitization Internal Standard “Spike” Assay Objectives Research, Development, or Process Budget Throughput Mixtures and Single Compound Screening. Scalability Sensitivity Error Tolerance False Positives and False Negatives Chapter 5: Format Homogeneous and Heterogeneous Pros and Cons Heterogeneous Immobilization Direct and Indirect Second Mediators Second Antibodies Biotin-avidin Enzyme Reporter Systems Alkaline Phosphatase and Horseradish Peroxidas Substrates Agonists and Antagonists Competition In vitro and in vivo In Vitro Assays In Vivo Assays Whole Organisms Biological Material Isolated Tissues and Perfused Organs Cell Cultures Bacteria and Eukaryotes Cell Lines and Primary Cultures Adherent and Suspension Cultures In Vitro Assays Blood and Blood Fractions Sub-cellular Fractions RER and Liver Microsomes Chapter 6: Readout (Reporting Format) Colorimetric and Fluorometric Colorimetric Assays Simple Ultraviolet and Visible Light Absorption Chromatography Staining Chromogenic Substrates Fluorometric Assays Fluorogenic Enzyme Cleavage Substrates Fluorescence Quenching EDANS and Dabcyl Green Fluorescent Protein Factors That Can Perturb GFP Fluorescence pH Effects and GFP Dimerization Denaturation Organic Solvents and GFP Proteases and GFP Detergents and GFP Oxidizing and Reducing Agents and GFP Chaotropes Physical Characteristics of GFP Fluorescence Fluorescence Measurements Excitation Emission Molecular Extinction Coefficients Quantum Yield Stokes Shift Fluorescence Lifetime Energy Transfer Radiative (Trivial) Energy Transfer Radiationless Energy Transfer Common Problems in GFP Fluorescence Detection Autofluorescence Recognizing Autofluorescence Reducing Autofluorescence Photostability Photobleaching Photoisomerization Photoconversion Fluorescence Detection and GFP General Considerations Available Light Sources for GFP Excitation GFP Emission Spectra and Fluorescence Detection Systems The Role of Filter Selection Instrument Sensitivity Spectrophotometry Fluorimetry Fluorometric Plate Readers Flow Cytometry Native PAGE Electrophoresis Fluorescence Microscopy and Imaging Environment Sample Preparation and Maintenance Fluorescence Microscope Set-Up Types of Fluorescence Microscopy Wide-field Fluorescence Microscopy Confocal Microscopy Two Photon Excitation Microscopy (TPEM) Advanced Fluorescence Microscopy Techniques Fluorescence Recovery After Photobleaching (FRAP) Fluorescence Loss in Photobleaching (FLIP) Fluorescence Correlation Microscopy (FCM) Fluorescence Lifetime Imaging (FLIM) Fluorescence Resonance Energy Transfer (FRET) Microscopy Total Internal Reflection Fluorescence Microscopy (TIR-FM) Calibrations and Data Analysis Data Output from Spectrophotometers A280 Readings Wavelength Scans. Molar Extinction Coefficients (MEC’s) Data Output from Fluorimeters Data Output from Fluorometric Plate Readers Data Output from Flow Cytometry Data Output from Native PAGE Electrophoresis Data Output from Microscopy and Imaging Cameras Image Capture Image Analysis Image Storage Time-resolved Fluorometry LANCE DELFIA Fluorescence Polarization Chemiluminescence Electrochemiluminescence ALPHAScreen Aequorin Radiometric Radiolabeling Radioimmunoassay Scintillation Proximity Assay Biological Growth Special Growth Media Other Readouts Chapter 7: Validation Installation and Operation Qualification Instruments and Equipment Manufacturer’s Certification and Specifications Establish Log Books. Assay Optimization General Factorial Design Consider the Worst Case Range Sensitivity Upper and Lower Limit of Quantitation (ULOQ and LLOQ) Linearity Precision and Accuracy Other Variables Physical Plant Personnel Performance Qualification Instruments and Consumables Maintain Log Books Consumables Operation Re-certification Assay Parameters Data Certification Scale-up General Step-by-step Process Chapter 8: In Vitro Assays General Spectroscopy UV/vis Mass Spectroscopy. NMR Protein Assays Colorimetric Protein Assays Light Absorbance Dye Binding Metal Reduction Amino Acid Analysis. Protein Hydrolysis AAA via HPLC Enzyme Assays Enzyme Stability Proteases Matrix Metalloproteases Blood Clotting Enzymes Kinases and Phosphorylases Kinases Radiolabel Incorporation Phosphate Analogs Measurement of Kinase under Constant ATP Concentration Coupling Kinase to NAD/NADH Ratio Phosphorylases Enzyme SPA EMIT Binding Specificity Valence and Avidity Blocking and Washing8 Blocking Washing Antibodies and Immunoassays Antibodies Structures and Fragments Classes Storage and Stability Polyclonal and Monoclonal Ab Monospecific Ab8 Immunoassays8 Radioimmunoassays Competitive RIA8 Antigen Capture RIA8 Fluorescence Immunoassays ELISAs Antigen-capture, Indirect Format Antigen Capture, Direct Format Sandwich Double Sandwich Western Sample System Membrane Antibodies Detection. “Classical” Immunoassays Agglutination Agar Assays Fluorescence Quenching Fluorescence Polarization Scintillation Proximity Assay SPA on Beads Other Binding Assays Nucleic Acids General Sample Processing Polymerase Chain Reaction General Practical Considerations and Troubleshooting Quantitative PCR Reverse Transcriptase PCR (RTPCR) Hybridization Blots Chapter 9: Cell-based Assay Application Areas and Associated Assays Available General Pitfalls. Cell Number and Viability Cell Line Stability Controls Autofluorescence Non-specific Binding Probe Specificity Reagent Stability and Variability Dead Cell Fraction Fixed Cells Fixing Fluorescence In Situ Hybridization Immunofluorescence Assay Staining and Counter-staining Whole Cells Cell ELISAs and ELISpots Cell ELISAs ELISpots Cytotoxicity RBC Lysis Chromium Release Hepatotoxicity Toxicity P450 Induction Surface Binding and Membrane Transport Surface Binding Membrane Transport Trafficking and Translocation MAPK NF-6B GPCR ?-arrestin Pitfalls Nuclear Receptors Cell Quantitation Cell Quantitation via DNA Staining Hoechst Stain Vitality Membrane Permeability Vital Stains Cell Leakage Division Growth Mitotic Markers Motility Apoptosis General Chromatin Degradation Example of Multivariate Analysis of Apoptosis and Necrosis Mitochondrial Membrane Potential Cytoskeletal Changes Cytoplasmic Enzymes Cell Surface Markers Annexin Cellular Morphology SPA in Microplates. Fluorescence Flow Cytometry General Advantages and Limitations Advantages Limitations Fluorophore Selection Fluorescein Rhodamines Phycobiliproteins Cy Dyes Tandem Conjugates Controls Non-specific Binding Dead Cells Gating Applications Quantitative Microbiology Immunophenotyping Lysates Gene Expression Microarrays Chips Protein Expression (Gene Induction) Microscopy Stains Labels FISH and IFA Chapter 10: Automation Platforms Miniaturization Pros and Cons Scaling Robotics Conveyors and Workstations Integrated and Modular Examples Beckman Coulter CyBio Tecan Tomtec Liquid Handlers General Applications Replication Dry Spotting Delivery Size Syringes Ink Jets and Pens Calibration Cross-contamination Sources Estimation and Intervention Pipet Tips Positive Displacement Liquid Sensing Tips Cell Handling Culture Sample Prep Microplate Equipment Microplates Polymer Materials Surface Treatments, Coatings, and Grafts Seals and Stickers Plate Handlers Washers. Bar Codes 1 Cleaning and Maintenance Chapter 11: Emerging Technologies Image Analysis General Issues Practical Considerations Assay Format and Probe. Data Format Dynamic Range Depth of Field Cell Counting Illumination Filters and Dichroic Mirrors CCD Camera Multiplexing Multiplexed Assay Technologies The Need for Multiplexing Key Competitive Advantages of Multiplexed Assay Technologies Over Micro-Arrays Automation An Example Higher Density Formats Multiplexing in 96-well Plates 384-well Plates High Density Microplates Microarrays and Chips Commercial Microarrays Protein Microarrays Homemade Microarrays Microfluidics New Technologies Planar Waveguide Technology Flow Cytometry with Labeled Beads and Libraries Branched DNA Binding Assay Single Molecule Detection Virtual Screening Chapter 12: Information Management Data Analysis. Acquisition Signal-to-background and Signal-to-noise Ratios Precision and Accuracy Type 1 and Type 2 Error Random and Systematic Error Binning and Pooling Statistics Binding Constants Error and Standard Error Correlation Coefficient Standard Deviation Coefficient of Variance Z’ factor More Complicated Statistics Error Detection Error Correction Normalization and Data Condensing Data Standardization Statistical Analysis Chapter 13: Appendices Appendix 1: Resources for Detailed Protocols Organizations. Other Non-commercial Sites Journals and Other Commercial Publications Appendix 2: Checklist for General Assay Development Research Guide for General Assay Development General Sample Data Standard Checklist To Be Used for Any Assay Development Appendix 3: Microarray Assay Checklist Experiment Design Samples used, extract preparation and labeling Hybridization procedures and parameters Measurement data and specifications Array Design Appendix 4: Vendors Appendix 5: Examples of Troubleshooting In Vitro Early Development Sample Matrix pH Autofluorescence Problems with Miniaturization Liquid Handler Contamination Intermittent Robot Failure Late Stage Solubility Problem Cell-based Early Development Sample Matrix Toxicity Transient Expression Problems with Scale-up. Culture Contamination Chapter 14: References Table of Exhibits Exhibit 2.1 Common Causes of Non-reproducibility in Microplates Exhibit 3.1 General Assay Formats for Binding of Labeled Ligand. Exhibit 3.2 Common FRET Donor-quencher Pairs and their Minimum Förster .Radius (R0) Exhibit 3.2 Summary of Mainstream Technologies for Ion Channel Interrogation Exhibit 4.1 Major Assay Design Considerations and Potential Problems Addressed Exhibit 4.2 Common Causes of Assay Non-reproducibility Exhibit 4.3 Sample Standard Curve with Data Interpolation, Extrapolation Exhibit 4.4 Different Assay Emphasis at Various Drug Development Stages Exhibit 5.1 Comparison of Heterogeneous and Homogeneous Assay Formats Exhibit 5.2 Comparison of Adsorption and Covalent Coupling in Heterogeneous Assays Exhibit 5.3 Comparison of In Vitro and In Vivo Assays Exhibit 5.4 List of Common In Vivo Whole Animal Assays Exhibit 5.5 Comparison of Cell-based Assays and In Vitro Assays Exhibit 5.6 Common Cell Lines Used for Assays. Exhibit 5.7 Common Primary Cultures for Assay Exhibit 5.8 Common Coatings for Cell Culture Based Assays Exhibit 6.1 Advantages and Disadvantages of Fluorescent Formats for HTS Exhibit 6.2 General Factors Known to Perturb GFP Fluorescence Exhibit 6.3 Conditions Tolerated by Aequorea GFP (wt) Exhibit 6.4 Common Sources of Autofluorescence Exhibit 6.5 Comparative Analysis of Photobleaching in Select GFP Variants Exhibit 6.6 Examples of Light Sources for Fluorescence Excitation Exhibit 6.7 Examples of Laser Lines Used in the Excitation of Fluorescent Proteins Exhibit 6.8 Excitation Spectrum and Laser Lines Exhibit 6.9 Parameters to Consider when Choosing a CCD Camera Exhibit 6.10 Instrument Detection Limits for Fluorescein Exhibit 6.11 Advantages of Fluorimetry Exhibit 6.12 Factors to Consider when Making Fluorimetric Measurements Exhibit 6.13 Tips for Fluorescence Microplate Assays Exhibit 6.14 Some Variables Among Fluorescence Microplate Readers Exhibit 6.15 Sample Thickness and Microscopy Applications Exhibit 6.16 Advantages of Two-Photon Microscopy over Confocal Microscopy Exhibit 6.17 Requirements for Measurement of GFP Molar Extinction Coefficients Exhibit 6.18 Molar Extinction Coefficients for Selected GFP Variants Exhibit 6.19 Some Considerations when Measuring GFP Quantum Yields Exhibit 6.20 Confocal Imaging Software Features Exhibit 6.21 Typical DELFIA Assay Steps Exhibit 7.1 Example of Factorial Design Exhibit 7.2 Common Problems Arising from Automation of an Assay Exhibit 8.1 Comparison of Protein Assays Based on Light Absorbance. Exhibit 8.2 Comparison of Dye Binding Protein Assays. Exhibit 8.3 Comparison of Metal Reduction Protein Assays Exhibit 8.4 Binding Curves Plotted as Hyperbola and as Sigmoid. Exhibit 8.5 Useful Blocker Solutions Exhibit 8.6 Comparison of Different ELISA Formats Exhibit 8.7 Antigen Capture ELISAs Diagrammed, Direct and Indirect Formats. Exhibit 8.8 Sandwich ELISA and Competitive RIA Diagrammed Exhibit 8.9 Recommended Number of PCR Cycles by Template Amount Exhibit 8.10 PCR Primer Selection Criteria Exhibit 9.1 Cell-based Assay Application Areas and Associated Assays Available Exhibit 9.2 Common Pitfalls in Cell-based Assays Exhibit 9.3 Minimal Controls Required for Cell-based Assays. Exhibit 9.4 Troubleshooting FISH and IFA Exhibit 9.5 Measurable Biological Endpoints for Whole Cell Assays Exhibit 9.6 Sample ELISpot Protocol Exhibit 9.7 Suitable P450 Substrates Exhibit 9.8 Classical Inducers for Rat and Human Plated Hepatocytes Exhibit 9.9 Assayable Parameters for Vitality Exhibit 9.10 Assayable Markers for Apoptosis Exhibit 9.11 Common Applications for Flow Cytometry Assay Exhibit 9.12 Dye Selection for Flow Cytometry Exhibit 9.13 Controls Required for Flow Cytometry Exhibit 11.1 Assays Suitable for Development Using HCS Exhibit 11.2 Multiplexed Assay Concept Exhibit 11.3 Comparison of Different Multiplexed Assay Technologies Exhibit 12.1 Examples of Kinetic Constants for Fast Enzymes Exhibit 12.2 Ordered Steps for Quality Control of UHT Data

Summary:

Assays are procedures for detection or quantitation of a specific material within a sample that may contain other interfering materials. In biopharma, the measured material, also called analyte, is usually a drug or drug lead compound, a protein, a gene or gene product, or a particular impurity or metabolite. Some assays, called multivariate or multiplex, simultaneously report on the presence of more than one material. When it is necessary to treat the sample matrix to facilitate analysis, concentrating the analyte and removing interfering materials, care must be taken to determine the recovery efficiency of the pre-treatment, such as by including an internal standard. While it is theoretically possible to develop an assay with both high sample throughput and high information content, usually one of these is optimized at the expense of the other. Highthroughput assays are commonly performed as an early screen, before investing in a more definitive high-content procedure. The former primary assays are generally faster and less expensive, and are often more prone to inaccuracies, compared to the latter secondary assays. There are many other illustrative ways to divide assays into two or more types. One is the division between in vitro and in vivo assays. Both are useful for biopharma assays. In drug discovery, in vitro procedures exhibit properties superior for primary assays, and in vivo assays often make better secondary screens. Cell-based assays fill an important gap between purely in vitro and in vivo assays, and offer intermediate levels of cost, throughput, and information. When faced with a choice between either in vitro or in vivo assay formats, consider the goals of the assay program. Generate the required data in the most efficient way possible. Another division of assay technologies is between homogeneous and heterogeneous assays. Heterogeneous assays include a step for immobilization of the analyte, a reporter, or both. This capture step increases sensitivity by driving biochemical equilibria toward completion. But, like many means of signal amplification, it can also increase background. Homogeneous assays are performed without practical separation of the analyte from all other assay reagents. In addition to lower background, they usually offer higher throughput, compared to heterogeneous assays. Assays may be performed at standard scale, or at various levels of miniaturization. Miniaturization, using microplate and array formats, provides savings in some reagents and sometimes offers increased throughput. Expressed the other way, increased throughput generally requires some factor of miniaturization. Sometimes assays directly measure the analyte according to its inherent properties. This includes the spectral properties of chemical compounds, and the ability of organisms to grow in a particular defined medium. More often an assay employs a series or cascade of targets and secondary materials that interact with the analyte and provide a robust signal. Common forms of signaling readout include radioactivity, color, fluorescence and other light-generating processes. There are some aspects of assay development that are universally important for success. These include precision, optimization, standards, and data analysis. Other critical factors, such as scale, throughput, and accuracy often depend on requirements imposed by the four main aspects listed above. Proper validation of an assay, especially in GMP environments, requires appropriate development, with attention to all of these factors. When high-throughout is required, it is usually best to develop an assay at low-throughput and then migrate to an appropriate high-throughout platform. Simultaneously, the assay instrumentation may be tested with water or recycled consumables. Because of the resource drain created by testing a large set of samples for full qualification of such a system, high-throughout platforms are often only partly validated before beginning screening, with complete validation occurring as part of the ongoing assay operation. Different analytes pose characteristic problems in assay development. For enzymes, the main problem is usually the efficiency and stability of the analyte under assay conditions, which must be carefully optimized. For binding measurements, the problem is background or non-specific binding, which is usually addressed via blocking. Antibodies (Ab) are convenient receptors used in many assays, especially immunoassays. Their limitations arise from the many different subtypes and varieties of Ab. DNA is a robust analyte, but the most sensitive assays for nucleic acids exhibit poor accuracy.