BIOASSAY

NOOR WIJAYAHADI
• Bioassay = biological assay
 a type of scientific experiment
 essential in new drugs development
& monitoring pollutants
• Measure the effects of a substance on a living
organism.
•  qualitative or quantitative
• Quantitative bioassays are typically analyzed using
the methods of biostatistics
 Definition
– Estimation of the conc / potency of a substance
by measuring its biological response in living
systems
– i.e.Observation of pharmacological effects on
– [1] living tissues, or cells
– [2] microorganisms
– [3] animals
 Bioassays/Assays
Whole animals
Isolated organs of vertebrates
Lower organisms e.g. fungi, bacteria, insects,
molluscs, lower plants, etc.
Cultured cells such as cancer cells and tissues of
human or animal organs
Isolated sub-cellular systems, such as enzymes,
receptors, etc
The use of bioassays include:
• measurement of the pharmacological activity of new or
chemically undefined substances
• investigation of the function of endogenous mediators
• determination of the side-effect profile, including the
degree of drug toxicity
• measurement of the concentration of known
substances (alternatives to the use of whole animals
have made this use obsolete)
• assessing the amount of pollutants being released by a
particular source, such as wastewater or urban runoff.
 Drug discovery
• Targets: New and Established
– Established targets  are those for which there is a
good scientific understanding
– new targets  are all those targets that are not
"established targets”
• Process  Screening and Design
 Diseases- Molecular Basis
 Over- and under-expression of catalytic proteins
(enzymes)
 Toxins produced by microorganisms
 Viruses (wild DNA/molecular organisms) cause
cancers, AIDS, influenza etc.
 Mutation in DNA
 Congenital diseases due to genetic malfunctions
 Oxidation of biomolecules (proteins, carbohydrates,
lipids, nucleic acid), degenerative diseases and ageing
 Deficiency of essential elements, vitamin, nutrients etc.
I
 Principles of Bioassay
• Active principle to be assayed should show the same
measured response in all animal species
• The degree of pharmacological response produced should
be reproducible under identical conditions [Eg Adrenaline
shows same rise in BP in the same species under identical
cond: wt, age, sex, strain etc]
• The reference standard must owe its activity to the
principle for which the sample is being bioassayed
• Activity assayed should be the activity of interest
• Individual variations must be minimised / accounted for
• Bioassay might measure a diff aspect of the same
substance compared to chemical assay [Eg testosterone &
metabolites
 Types of Bioassays?
 In Silico Screenings
Non- physiological Assays
Biochemical or Mechanisms-Based Assays
In Vitro Assays
Cell based Bioassays
Tissue based Bioassays
In Vivo Bioassays
Animal-based Assays/Preclinical Studies
Human trial/Clinical Trials
 Broad Categories of Bioassays

Virtual Screenings
Primary Bioassays
Secondary Bioassays
Preclinical Trials
Clinical Trials
 Virtual Screenings

 Target Selection
 Data Mining (Chemical space of over 1060
conceivable compounds)
 Screening of Libraries of Compounds Virtually
 lead optimization
 Prediction of Structure-Activity Relationships
 Primary “Bioassay/Assays”
Screenings

Non- physiological Assays

Biochemical or Mechanism-Based Assays
In Vitro Assays
Microorganism-based bioassays
Cell-based Bioassays
Tissue-based Bioassays
 Examples of Primary Assays
 Antioxidant Assays
Enzyme Inhibition Assays
Cytotoxicty Bioassays
Anti-cancer Bioassays (Cancer Cell Lines)
Brine Shrimp Lethality Bioassays
In Vitro Antiparasitic Bioassays
Anti-bacterial Bioassays
Antifungal Bioassays
Insecticidal Bioassays
Phytotoxicity Bioassays
Etc.
 Salient Features of Primary Bioassay
Screenings
 Predictive Potential
General in nature
Tolerant of impurities
Unbiased
High-throughput
Reproducible
Fast
Cost-effective
Compatible with DMSO
 Hit Rate of Primary Bioassay
Screenings

 A hit rate of 1% or less is generally considered

a reasonable
False positive are acceptable
False negative are discouraged
 Secondary Bioassays

In Vivo Bioassays

Animal-based Assays/Preclinical Studies
 Predicting Drug Like Behavior- Lipinski “Rule of
Five”
• Molecular weight about 500 a. m. u. (Optimum 350)

• Number of hydrogen bond accepter ~ 10 (Optimum 5)

• Number of hydrogen bond donor ~ 5 (Optimum 2)

• Number of rotatable bonds ~5 (Conformational Flexibility)

• 1-Octanol/water partition coefficient between 2-4 range

 In Vitro Bioassays

 In Vitro: In experimental situation outside the

organisms. Biological or chemical work done in
the test tube( in vitro is Latin for “in glass”) rather
than in living systems
Examples include antifungal, antibacterial,
organ-based assays, cellular assays, etc
 Examples of In Vitro Bioassay
 Activity Assays
DPPH assay
Xanthine oxidase inhibition assays
Superoxide scavenging assay
Antiglycation assay
 Bioassays (cell-based)
DNA Level
Protein Level
RNA Level
Immunology assay
 Toxicity Assays
MTT assay
Cancer cell line assays
 In Vivo Screenings or Pharmacological
Screenings

 In Vivo: Test performed in a living system such

as antidiabetic assays, CNS assays,
antihypertensive assays, etc.
 Examples of In Vivo Bioassays

 Animal Toxicity
Acute toxicity
Chronic toxicity
 Animals Study
Animal model with induced disease
Animal model with induced injury
 Pre-Clinical Trials
 Clinical Trials
 High-throughput Assays

 The process of finding a new drug against a

chosen target for a particular disease usually
involves high-throughput screening (HTS),
wherein large libraries of chemicals are tested for
their ability to modify the target.
 HIGH-THROUGHPUT BIOLOGICAL
SCREENINGS

• Development of straight-forward in-vitro

biological assays (enzyme-based, cellular and
microbiological assays) into automated high-
throughput screens (HTS).
• Rapid assays of thousands or hundreds of
thousands of compounds (upto 100,000 samples
per day).
• Specifically suitable for the isolation of bioactive
constituents from complex plant extracts.
High-throughput Screening Strategy
for Enzyme Inhibition Assays
% Inhibition = [(E-S)/E]  100
Enzyme + Buffer
E = Activity of enzyme without test material
+ Potential inhibitor
S = Activity of enzyme with test material

Substrate

Incubation
Measurement of absorbance
96-well plate

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Blood collection from the saphenous vein
Blood collection from the saphenous vein
Blood collection from the saphenous vein
making a mouse-sized tourniquet
Blood collection from the dorsal pedal vein
Retro-orbital blood collection
Proper positioning of mouse for blood collection from the jugular vein
blood collection from the jugular vein