Ge Wu

Assay Development

Fundamentals and Practices

• Gebundenes Buch

Produktbeschreibung

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. After discussing the basics, it explains the treatment of assays for purified protein, cell based assays, and high throughput screening. It includes good practices and case studies. This is a premier resource for biotechnologists, analytical chemists, biochemists, medicinal chemists, toxicologists, and others, and an excellent text for professors preparing students to enter the biotechnology and pharmaceutical industries.

Produktdetails

• Verlag: Wiley & Sons
• 1. Auflage
• Seitenzahl: 448
• Erscheinungstermin: 26. April 2010
• Englisch
• Abmessung: 240mm x 161mm x 29mm
• Gewicht: 769g
• ISBN-13: 9780470191156
• ISBN-10: 0470191155
• Artikelnr.: 23594978

Autorenporträt

Ge Wu, PhD, is Director of Assay Development at Iris International. He obtained his doctorate in biochemistry from UCLA, performed postdoctoral work at Harvard Medical School, and was a member of the faculty at Cornell University Medical College. Prior to joining Iris International, he worked at Merck & Co., Caliper Technologies, Amphora Discovery, and FivePrime Therapeutics. He is the founding Director of Assay Development and Screening at FivePrime Therapeutics. Dr. Wu is a leading figure in assay development and screening in biopharmaceutical discovery using primary cell assay and the author of an acclaimed review in this subject.

Inhaltsangabe

Preface. Chapter 1 Introduction to assay development. 1.1 Assay and
Bioassay. 1.2 Drug discovery process and the role of assays in the process.
1.3 Bioassay development. 1.4 Bioassay Classifications. Chapter 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 (TRF)
measurement.. 2.6 Fluorescence resonance energy transfer (FRET) and
TR-FRET. 2.7 Fluorescence quenching. 2.8 Fluorescence polarization. 2.9
Radioactivity measurement. 2.10 Evaluating and selecting an instrumental
method for bioassay. Chapter 3 Fundamental principles of assay with
isolated proteins. 3.1 Chemical potential, equilibrium, and kinetics. 3.2
Protein binding studies at equilibrium. 3.3 Kinetic studies of protein
binding process. 3.4 Enzyme kinetics. 3.5 Inhibition of protein function.
3.6 Assay development with isolated proteins. Chapter 4 Separation-based
techniques in bioassays. 4.1 Washing to remove impurity on solid support.
4.2 Organic extraction of hydrophobic molecules. 4.3 Centrifugation to
remove dense particles. 4.4 Membrane filtration. 4.5 Liquid chromatography.
4.6 Electrophoresis. Chapter 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 SPATM and FlashplateTM in binding
studies. 5.4 Application of FP assays 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. Chapter 6 Functional assays with isolated proteases. 6.1
Introduction to proteases and their substrates. 6.2 The function of
proteases and their role drug discovery. 6.3 Protease assays. 6.4 Protease
substrate profiling. 6.5 Protease inhibitors. 6.6 Assay development for
caspases with fluorogenic substrate.. 6.7 Assay development for
Carboxypeptidase U.. Chapter 7 Functional assays with isolated 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 the
detection of the 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 measurement of both product and substrate simultaneously. 7.9 An
example of a kinase assay development in HTRF format.. Chapter 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 The concept of affinity and efficacy in cell-based assays. 8.4
Development of cell-based assays. Chapter 9 Assays with ion channels. 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.
Chapter 10 Assays with G protein-coupled receptors. 10.1 Introduction to
GPCRs and G proteins. 10.2. GPCR activation and signal transduction. 10.3
Strategies of GPCR assay development. 10.4 GPCR assays by measuring the
extent of GTP binding to G?. 10.5 GPCR assays based on measurement of cAMP.
10.6 GPCR assays based on the measurement of intracellular inositol
phospholipids. 10.7 GPCR assays based on the measurement of intracellular
Ca2+. 10.8. GPCR assays based on the measurement of MAPK activity. 10.9.
GPCR assays with reporter gene. 10.10 GPCR assays by monitoring events
leading to GPCR internalization. Chapter 11 Assays based on the integrated
system properties of cells. 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. Chapter 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 image-based cellular high content screening. Chapter 13
High throughput screening. 13.1 Introduction to high throughput screening.
13.2 Molecular or cellular target 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.
Chapter 14 A case study: The development of a microfluidic-based kinase
assay platform. 14.1 Background of microfluidic technology and its
application in bioassays. 14.2 The original Caliper?s mobility shift kinase
assay format. 14.3 Realizing the flaws in Caliper?s original kinase assay
format. 14.4 Searching for alternative kinase assay methods. 14.5 The
development of the off-chip kinase assay format. 14.6 Current stage of
microfluidic technology in bioassays. 14.7 Appendix: A poster presented in
2002 Society for Biomolecular Screening annual meeting.