Bioassay Techniques for Drug Development

by ;
Edition: 1st
Format: Hardcover
Pub. Date: 2001-10-04
Publisher(s): CRC Press
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Summary

The goal of an activity-directed isolation process is to isolate bioactive compounds which may provide structural leads of therapeutic importance. Whereas the traditional process of drug development is long and expensive, simple and rapid bioassays can serve as the starting point for drug discovery. This book presents a range of "bench top" bioassay techniques useful for natural product and pharmaceutical chemists involved in drug discovery and pharmacognosy. The contents detail a number of enzyme-based assays, cell-based functional bioassays and receptor radioligand binding assays along with detailed descriptions of each type. The majority of these bioassays are presented in a step-by-step format, so they could even be implemented by technical personnel with little background in microbiology, biochemistry or pharmacology.

Table of Contents

Preface xi
General Introduction 1(6)
Bench-Top and Primary Bioassay Screening
7(100)
Toxicity Assays
9(5)
Brine-shrimp lethality assay
9(1)
Brine-shrimp microwell cytotoxicity assay
10(2)
Crown gall tumor inhibition assay (potato disc antitumor assay)
12(2)
Animal toxicity assay
14(1)
Antimicrobial Assays
14(11)
Antibacterial assays
16(1)
Agar diffusion assay
16(1)
Agar dilution assay
17(1)
Microtitre plate method
18(1)
Direct bioautobiography method
19(3)
Antifungal assays
22(1)
Agar tube dilution assay
22(2)
Direct bioautobiography method
24(1)
Antiviral and Anticancer Assays
25(14)
Anti-HIV assay
25(2)
Anticancer assays
27(1)
In vitro anticancer screening
28(9)
HCT cytotoxicity assay
37(2)
Antimitotic Assay
39(2)
Antimitotic assay using sea urchin eggs
39(2)
Genotoxicity Assays
41(7)
SOS chromtest for genotoxicity
41(3)
Ames test for detecting carcinogens and mutagens
44(3)
Antiphage activity assay
47(1)
Assays for Control of Tropical Diseases
48(17)
Antimalarial assays
48(1)
96 well plate in vitro schizonticidal assay
49(1)
Suppressive test of blood schizonticidal action (Peter's test)
50(1)
Rane test of blood schizonticidal activity
51(1)
Larvicidal assay
51(1)
Molluscicidal assay
52(1)
Piscicidal assay
53(1)
Amoebicidal assay
54(5)
Cercaricidal assay
59(1)
Leishmanicidic activity
60(1)
In vitro Leishmanicidal assay (promastigotes)
60(2)
In vitro Leishmanicidal assay (amastigotes)
62(2)
Leishmanicidal activity in vivo
64(1)
Assays for Agrochemicals
65(9)
Lemna minor for phytotoxicity and growth-stimulating assay
65(2)
Contact toxicity insecticidal assay
67(1)
Insect antifeedant assays
68(1)
Leaf-choice assay
68(1)
Artificial diet feeding bioassay
69(3)
Nematicidal assay
72(2)
Hepatotoxicity Assays
74(8)
Antihepatotoxic activity using carbon tetrachloride-induced cytotoxicity
74(2)
In vivo hepatoprotective assay
76(2)
Antihepatotoxic activity using galactosamine induced cytotoxicity
78(4)
Hypoglycemic/Antidiabetic Activity Assays
82(5)
Antidiabetic activity assay on normal and alloxan-diabetic rabbits
82(2)
Antidiabetic activity assay (GOD-PAP method)
84(1)
Hypoglycemic activity assay
85(2)
Diuretic Activity Assay
87(1)
Anthelmintic Activity Assays
88(2)
In vitro anthelmintic assay
88(2)
In vivo anthelmintic assay
90(1)
Antifertility/Anti-implantation Assays
90(2)
Antifertility activity
90(1)
Anti-implantation activity
91(1)
In Vitro Assay for Platelet Aggregation
92(2)
Anti-inflammatory Assay
94(1)
Rat paw edema assay
94(1)
Immunomodulating Assay
95(1)
Hemolytic plaque assay
95(1)
Antiepileptic (Anticonvulsant) Assay
96(4)
Pilocarpine model
97(3)
Analgesic Assays
100(1)
Writhing syndrome method
100(1)
Hot plate method
100(1)
Gastroprotective/Antiulcer Assays
101(2)
Gastroprotective assay
101(1)
Anti-ulcerogenic assay-1
102(1)
Anti-ulcerogenic assay-2
103(1)
Radiolabelling Bioassays
103(3)
Mitogenic activity assay
103(3)
Anti-emetic Assay
106(1)
High-Throughput Screening
107(112)
Introduction
109(5)
Characteristics of an ideal screening assay for screening natural product extracts
109(1)
Biochemical or cell based assays
110(1)
Assay format
110(1)
Reagent source
111(1)
Assay signal-to-noise ratio
112(1)
Assay incubation
112(1)
Assay manipulations
113(1)
DMSO compatibility
113(1)
Screening source material compatibility
113(1)
Enzyme Assays
114(38)
Introduction
114(1)
Considerations for development of enzyme assays
114(1)
Selection of enzyme source
115(1)
Selection of enzyme concentration
116(1)
Selection of substrate and substrate concentration
116(5)
Selection of cofactor concentrations
121(1)
Selection of assay endpoint
122(1)
Selection of assay pH
122(1)
Selection of assay temperature
123(1)
DMSO compatibility
123(1)
Extract compatibility
123(1)
Inhibitor profiles
123(1)
Competitive inhibition
124(1)
Uncompetitive inhibition
125(1)
Noncompetitive inhibition
125(1)
Development of a high throughput enzyme assay for natural product screening: an example
126(7)
Some common in vitro enzyme-based bioassays
133(1)
Protease inhibition assays
133(4)
Tyrosinase inhibition assay
137(2)
Hyaluronidase inhibition assay
139(3)
Acetylcholinesterase inhibition assay
142(1)
Spectrophotometric assay
143(2)
β-Lactamase inhibition assay
145(1)
Spectrophotometric assay
146(2)
5-Lipoxygenase inhibition assay
148(1)
Spectrophotometric assay
149(3)
Cell-based Receptor Functional Assays
152(27)
Introduction
152(1)
Utility of receptor functional assays
152(1)
Functional assays for G-protein coupled receptors
152(1)
Gs and Gi coupled receptor signal transduction pathways
152(3)
High throughput functional assays for Gs and Gi coupled receptors
155(1)
Common cell-based receptor functional assays
155(1)
SPA cAMP assay
155(1)
FlashPlate cAMP assay
156(1)
ELISA-based cAMP assays
157(3)
cAMP response element reporter gene assay
160(1)
Gq-coupled receptor signal transduction pathways
161(1)
Introduction
161(3)
High throughput assays for Gq-coupled receptors
164(1)
High throughput functional assays for ligand-gated ion channels
165(2)
Receptor protein tyrosine kinase functional assays
167(1)
Introduction
167(1)
RPTK-associated signal transduction pathways
167(2)
High throughput functional assays for RPTK'S
169(1)
Intracellular calcium assay
169(1)
Proliferation assays
170(3)
Cytokine receptor functional assays
173(1)
Cytokine receptor signal transduction pathways
173(1)
Functional assays for cytokine receptors
173(1)
Mixed receptor functional assay
174(1)
Melanophore functional receptor assay technology
174(4)
Receptor selection and amplification functional receptor assay technology
178(1)
Radioligand Binding Assays
179(26)
Introduction
179(1)
Important considerations for developing radioligand binding assays
180(1)
Receptor source and assay concentration
180(1)
Nonrecombinant human cell lines
180(1)
Membrane preparations from nonrecombinant cells and tissues
181(1)
Recombinant receptor containing cells or membranes
181(1)
Selection of radioligand and assay concentration
182(2)
Selection of assay incubation conditions
184(1)
Selection of assay buffer
184(1)
Selection of assay volume
184(1)
Selection of a methodology for separation of free and bound radioligand
185(1)
Steps in developing and validating radioligand binding
185(1)
Steps in developing and validating radioligand binding assays
185(1)
Cell membrane linearity evaluation
186(1)
Association and dissociation time courses
187(5)
Saturation isotherm evaluation
192(2)
Competition studies
194(4)
Alternative radioligand binding assays
198(1)
FlashPlate assays
198(2)
Scintillation proximity assays
200(1)
ScintiStrip plate assays
201(2)
Fluorescence based receptor binding
203(1)
Time resolved fluorescence
203(1)
Fluorescence polarization
203(2)
References
205(14)
Index
219

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