Assay development : fundamentals and practices / Ge Wu.

Format:

Book

Author/Creator:

Wu, Ge, 1962-

Language:

English

Subjects (All):

Biological assay.

Aquatic biology.

Physical Description:

1 online resource (425 p.)

Edition:

1st ed.

Place of Publication:

Hoboken, N.J. : Wiley, c2010.

Language Note:

English

Summary:

Essential principles and practice of assay development The first comprehensive, integrated treatment of the subject, Assay Development: Fundamentals and Practices covers the essentials and techniques involved in carrying out an assay project in either a biotechnology/drug discovery setting or a platform setting. Rather than attempting comprehensive coverage of all assay development technologies, the book introduces the most widely used assay development technologies and illustrates the art of assay development through a few commonly encountered biological targets in assay development (e.g., proteases, kinases, ion channels, and G protein-coupled receptors). Just enough biological background for these biological targets is provided so that the reader can follow the logics of assay development. Chapters discuss: * The basics of assay development, including foundational concepts and applications * Commonly used instrumental methods for both biochemical assays and cell-based assays * Assay strategies for protein binding and enzymatic activity * Cell-based assays * High-throughput screening An in-depth study of the now popular Caliper's off-chip kinase assay provides an instructive, real-world example of the assay development process.

Contents:

Intro

ASSAY DEVELOPMENT

CONTENTS

FOREWORD

PREFACE

ACKNOWLEDGMENTS

ABBREVIATIONS

1 INTRODUCTION TO ASSAY DEVELOPMENT

1.1 Assay and Bioassay

1.2 Drug Discovery Process and Role of Assays in the Process

1.3 Bioassay Development

1.4 Bioassay Classifications

Useful Websites

Bibliography

2 MEASUREMENT AND INSTRUMENTATION

2.1 Measurement and Perturbation

2.2 Common Instrumental Methods and Instrument Components

2.3 Molecular Absorption Measurements

2.4 Molecular Luminescence Measurements

2.5 Luminescence Lifetime Measurement and Time-Resolved Fluorescence Measurement

2.6 Fluorescence Resonance Energy Transfer (FRET) and Time-Resolved-FRET

2.7 Fluorescence Quenching

2.8 Fluorescence Polarization (FP)

2.9 Radioactivity Measurement

2.10 Evaluating and Selecting an Instrumental Method for Bioassay

3 FUNDAMENTAL PRINCIPLES OF ASSAY DEVELOPMENT WITH ISOLATED PROTEINS

3.1 Chemical Potential, Equilibrium, and Kinetics

3.2 Protein Binding Studies at Equilibrium

3.3 Kinetic Studies of the Protein Binding Process

3.4 Enzyme Kinetics

3.5 Inhibition of Protein Function

3.6 Assay Development with Isolated Proteins

4 SEPARATION-BASED TECHNIQUES IN BIOASSAYS

4.1 Washing Solid Supports to Remove Impurities

4.2 Organic Solvent Extraction of Hydrophobic Molecules

4.3 Centrifugation to Remove Dense Particles

4.4 Membrane Filtration

4.5 Liquid Chromatography

4.6 Electrophoresis

5 GENERAL PROTEIN BINDING ASSAY FORMATS

5.1 Equilibrium Dialysis

5.2 Competitive Binding Assays with Radioactive or Other Labeled Ligands

5.3 Application of SPA and FlashPlate in Binding Studies

5.4 Application of Fluorescence Polarization (FP) in Binding Studies.

5.5 Application of FRET Assays in Binding Studies

5.6 Application of ELISA in Binding Studies

5.7 Surface Plasmon Resonance (SPR) Technology and Its Application in Binding Studies

5.8 Application of Label-Free Technologies in Binding Studies

6 FUNCTIONAL ASSAYS WITH ISOLATED PROTEASES

6.1 Introduction to Proteases and Their Substrates

6.2 Function of Proteases and Their Role in Drug Discovery

6.3 Protease Assays

6.4 Protease Substrate Profiling

6.5 Protease Inhibitors

6.6 Assay Development for Caspases with a Fluorogenic Substrate

6.7 Assay Development for Carboxypeptidase U (EC 3.4.17.20)

Useful Websites for Proteases

7 FUNCTIONAL ASSAYS FOR PROTEIN KINASES

7.1 Introduction to Protein Kinases

7.2 Substrates for In Vitro Kinase Assays

7.3 Kinase Assay Development Strategies

7.4 Kinase Assay Based on Detection of Phosphorylated Product

7.5 Kinase Assays by Measuring the Generation of ADP

7.6 Kinase Assays by Measuring the Depletion of ATP

7.7 Kinase Assays by Measuring the Depletion of Peptide Substrate

7.8 Kinase Assays by Simultaneous Measurement of Both Product and Substrate

7.9 Example of a Kinase Assay Development in HTRF Format

8 FUNDAMENTAL PRINCIPLES OF CELL-BASED ASSAYS

8.1 Cell Signaling, Signal Transduction, and Cellular Responses

8.2 General Approaches in Cell-Based Assays

8.3 Concept of Affinity and Efficacy in Cell-Based Assays

8.4 Development of Cell-Based Assays

9 FUNCTIONAL ION CHANNEL ASSAYS

9.1 Introduction to Ion Channels

9.2 Strategies for Ion Channel Assays

9.3 Electrophysiological Methods

9.4 Ion Flux Methods

9.5 Membrane Potential Sensing Methods

9.6 Selecting Suitable Assays for Ion Channel Studies.

Useful Websites and Vendors

10 ASSAYS WITH GPCRs

10.1 Introduction to GPCRs and G Proteins

10.2 G Protein-Coupled Receptor Activation and Signal Transduction

10.3 Strategies of GPCR Assay Development

10.4 G Protein-Coupled Receptor Assays by Measuring the Extent of GTP Binding to G(α)

10.5 G Protein-Coupled Receptor Assays Based on Measurement of cAMP

10.6 G Protein-Coupled Receptor Assays Based on Measurement of Intracellular Inositol Phospholipids

10.7 G Protein-Coupled Receptor Assays Based on Measurement of Intracellular Ca(2+)

10.8 G Protein-Coupled Receptor Assays Based on Measurement of MAPK Activity

10.9 G Protein-Coupled Receptor Assays with Reporter Gene

10.10 G Protein-Coupled Receptor Assays by Monitoring Events Leading to GPCR Internalization

11 ASSAYS BASED ON INTEGRATED CELL SYSTEM PROPERTIES

11.1 Cell Viability, Proliferation, and Cytotoxicity Assays

11.2 Measurement of Extracellular Indicators of Cellular Metabolism

11.3 Measurement of Cell's Effect on Electrical Impedance

11.4 Measurement of Protein Secretion from Cells

11.5 Measurement of Discoloration of Melanophore Cells

11.6 Measurement of Cell Motility

12 HIGH-CONTENT CELL-BASED ASSAY WITH OPTICAL IMAGING TECHNIQUES

12.1 Sample Preparation

12.2 Cellular Image Collection

12.3 Image Abstraction, Analysis, and Data Management

12.4 Applications of iCHCS

13 HIGH-THROUGHPUT SCREENING

13.1 Introduction

13.2 Molecular or Cellular Targets and Assay Development

13.3 Compound Library Management

13.4 Hardware Module

13.5 Software Module

13.6 HTS Operation Management

13.7 Building an HTS Operation for Biopharmaceutical Discovery.

13.8 Quality Control and Data Analysis in Primary Screening

14 CASE STUDY: DEVELOPMENT OF A MICROFLUIDIC-BASED KINASE ASSAY PLATFORM

14.1 Background of Microfluidic Technology and Its Application in Bioassays

14.2 The Original Mobility Shift Kinase Assay Format

14.3 Realizing the Flaws in the Original Kinase Assay Format

14.4 Searching for Alternative Kinase Assay Methods

14.5 Development of the Off-Chip Kinase Assay Format

14.6 Current Stage of Microfluidic Technology in Bioassays

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

INDEX.

Notes:

Bibliographic Level Mode of Issuance: Monograph

Includes bibliographical references and index.

Description based on metadata supplied by the publisher and other sources.

ISBN:

9786612656514

9781282656512

1282656511

9780470583128

0470583126

9780470583111

0470583118

OCLC:

644162814