Printer Friendly Printed from http://www.researchandmarkets.com/reports/227827 A Guide to HTS Assay Development Description: Assays for high-throughput screening (HTS) are growing explosively. Many exciting new technologies have recently been introduced, while several existing technologies have catapulted out of academic labs and into common use. At the same time, some older techniques have "hit the wall" and fallen out of favor, as others have surmounted the challenges of modernization and remained competitive. In an expanding effort to fill their pipelines with new clinical candidates, the drug industry is struggling with ever-increasing expenditure for discovery of new compounds. For most biotechnology and pharmaceutical organizations, HTS represents the beginning of the drug development pipeline. Improvements in this early stage are extremely cost-effective, since they eliminate time and money wasted on poor leads. Thus, utilizing the appropriate assay to efficiently identify hits, and especially identification of high-quality hits, is crucial to drug development economy. Scope of the Guide This Guide thoroughly evaluates the capabilities, strengths, weaknesses, and expectations of leading assays and assay technologies for high-throughput screening. The organization of the Guide is tailored to reflect the importance of both assay format and target class. Questions Answered: - When are cheaper assays better? - What scientific and business factors justify increased cost for an assay? - If time is money, how can slower assays be more economical than fast ones? - When is it OK to perform a screen before validating an HTS assay? - What are the real benefits of miniaturization? Do these benefits extend beyond 384-well plates to 1536-well plates, or even further? - What are the available choices for HTS format for my target of interest? What is the best choice for my particular goals? Target Audience: The following professionals involved in drug discovery, screening, lead generation, and assay technology will greatly benefit from this Guide: Directors, Lab Managers, Group Leaders, Senior Scientists, Principal Scientists, Project Managers, Heads of Research & Development, and more Contents: 1 Introduction Executive Summary New Paradigms New Technology The Three-Way Compromise Economy - The Fourth Variable Validation Planning Flexibility Primary and Secondary Screening Data Analysis Assay Structure Format GPCR and Ion Channels Platform Purpose and Scope Exclusions Trends 2 Planning An HTS Program General Program Goals Scientific and Business Goals Assay Objectives Common Problems and Pitfalls Special Considerations for Microarrays Assay Design and Optimization Instruments and Equipment Sampling and Dilutions Mixing and Multiplexing Sample Mixtures Target Mixtures Target Multiplexing Primary and Secondary Assays Assay Optimization Serial Dilutions Full Factorial Design Standards Standard Curves Curve Modeling Checklist for Assay Development Universal Considerations Precision Optimal Reagent Amounts Data Analysis Assay Format Homogeneous and Heterogenous Multiplexing Reporting System Direct Indirect Agonists, Antagonists, Partial Agonists, and Competition Agonists and Antagonists Competition Data Format Colorimetrics Fluorescence Tramp Fluorescence Autofluorescence Chemiluminescence Other Formats References 3 Assay Structure (Format) Binding Specificity Background Blocking Washing Affinity, Valence, and Avidity Affinity Valence Avidity Detection Molecular Size Biochemical Function Labels Enzyme Labels RCA Fluorescent Label Methods Fluorescence Intensity Fluorescence Quenching and Fret Fluorescence Polarization Fluorescence Correlation Antibodies and Immunoassays Antibodies Polyclonal and Monoclonal Ab Hybridomas Practical Considerations Ab Labeling Immunoassays and Elisas Capture and Sandwich Elisas Mimotopes Standard Curves Ab Cross-Reactivity Cell Elisas and Elispots Cell Elisas Elispots Bead Assay Platforms Magnetic Beads Fmat Origen Xmap Alphascreen Other Other Binding Assays Scintillation Proximity Assay (SPA) Nucleic Acid Hybridization Gene Expression Analysis Purification and Amplification Probes and Labels for Nucleic Acid Detection Fluorescence Arrayplate Bdna Enzyme Assays General Assumptions Enzyme Stability Strategies Substrate Consumption Product Appearance Coupled Systems LDH Kinetics and Endpoints Enzyme Engineering Important Enzyme Targets Proteases Kinases and Phosphorylases Phosphorylation Assays ATP Consumption Other Important Enzymes DNA Elongation PCR and QPCR in HTS Licensing Modularity Principles of PCR Principles of QPCR Tips for QPCR QRTPCR Other Important HTS Assays ADME Solubility Absorption Caco2 PAMPA Biodistribution Drug Metabolism and Excretion References 4 Miniaturization And Automation Miniaturization General Advantages Disadvantages Management Microplates and Liquid Handlers Microplates Standards Tips Racks and Microtubes Miscellaneous and Extra-Small Wells Black, White, and Clear Coatings Common Microplate Problems Damage Warp Cleanliness Special Problems with 1536-Well Plates Microplate Equipment Seals Mats Stickers Iron-Ons Seal Validation Bar Codes Microplate Handlers Integrated or Modular Organization Linear Cylindrical Access and Security Liquid Handlers and Dispensers Variables Volume Calibration Fixed and Disposable Tips Nanoliter Measurement Pens Non-Contact Delivery Software Plate Washers Microplate Density One- and Two-Pin Models Flow Rate and Volume Cell Handling Cleaning and Maintenance Testing Arrays and Microarrays General Arrays Versus Microarrays cDNA Versus Oligonucleotides Quality Whole Genome Chips Manufacturing Non-Human Chips Homemade and Custom Arrays Substrates Oligo Sets Flow-Through Chips High-Throughput RNA Preparation Cell Disruption Purification of RNA RNA Stabilization Automation Commercial Kits for RNA Purification Microarray Data Analysis Errors Stepwise Analysis Background Gene Linkage Miame and Mage Miame Mage Arrayexpress Tips Validate Splice Variants Use Enough Buffer Reduce Ambient Light Hold Your Breath Temperature Controls Array Readers FI Alternatives to FI References 5 Cell-Based Assays General Important Controls Autofluorescence Cell Autofluorescence Sample Compound Fluorescence Non-Specific Binding Probe Specificity Reagent Stability and Variability Viability Applications Viability and Cytotoxicity Medium Acidification Metabolic Activity Plasma Membrane Integrity Apoptosis Protein Trafficking and Translocation Gpcr And ß-Arrestin Mapk Nuclear Receptors Nf-kB Toxicity P450 Nephrotoxicity Cardiac Arrhythmia Bone Marrow Strategies Fixing Transfect and Over-Express Target Protein Protein Engineering EBV Episomes Clone and Express Multiplexing Large-Scale Cell Culture Out-Sourcing Common Problems High Content Screening (HCS) General Development Cytometry and Cell Sorting Simple High-End Confocal Imaging Historical Introduction Arc and Laser Illumination Scanners Spinning Disks Others HCS Strategies General DNA Measurements Viability Nuclear Receptors Promiscuous Heterodimers Image Analysis General Hardware Software Algorithms Common Artifacts Lysates Lysis Buffers Filter-Based Binding Assays Protein Expression and Gene Induction Important Targets for Cell-Based Assays GPCR Strategies Ligand Binding Assays Membrane Preps Second Messengers Calcium Ip3 Camp Gtp Binding Reporter Genes Xenopus Melanophore Protein Trafficking ß-Arrestin Ion Channels Ion Flux Assays Patch-Clamp Automated Patch-Clamp Platforms Reporter Dyes Voltage Sensitive Dye Systems Membrane Binding Assays Automated Cell Culture Pitfalls Plating Uneven Suspension Culture Plate Surface Treatments Phenotype Characterization of Transfected Cell Lines Western Blot and Cytometry Handling and Cell Culture Conditions Viability Biology Fixing Emerging Platforms for Cell-Based Assays References 6 Information Management Data Acquisition Data Analysis Expectations Strategies Error Detection Error Correction Normalization and Data Condensing Data Standardization Statistical Analysis Binning and Pooling Statistics General Strategies Random and Systematic Error Random Error Systematic Error Bias Type 1 and Type 2 Error Sample Number (N) Signal-To-Noise and Signal-To-Background Ratios Signal-To-Noise Signal-To-Background Limit Of Detection Precision and Accuracy Standard Deviation Coefficient of Variance Resolution Residual Analysis Ordinary Least Squares Residual Analysis Z' Factor Software and Automated Data Analysis References 7 Emerging Technologies Ultraminiaturization Microfluidics Mems Non-Image-Based Hcs Protein Arrays Ab Arrays Substrates Tissue Arrays Evanescent Waves Single Molecule Detection High-Throughput Clinical Diagnostics Miscellaneous Quantum Dots Thermal Stability Bar Codes QTL Etags Ultrasonics Liquid Crystals Virtual Screening References 8 Appendices Abbreviations, Acronyms, and Definitions Common Equations Binding Constants Equilibrium Binding Binding Kinetics Inhibitors Enzyme Kinetics Information Sources Resources for Detailed Protocols Orgs Schools and Other Non-Commercial Sites Journals and Other Commercial Publications Checklist for HTS Assay Development Research Guide for General Assay Development General Sample Data Standard Checklist Miame Checklist for Microarrays Experiment Experimental Design Samples Used, Extract Preparation and Labeling Hybridization Procedures and Parameters Measurement Data and Specifications Array Design The Miame Checklist Experiment Design Samples Used, Extract Preparation and Labeling Hybridization Procedures and Parameters Measurement Data and Specifications Array Design Vendors References Ordering: Order Online - visit http://www.researchandmarkets.com/reports/227827 Order by Fax - using the order form below Order By Post - print the order form below and send to Research and Markets, Guinness Centre, Taylors Lane, Dublin 8, Ireland. 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