2. OVERVIEW
BIOASSAY – Definition/Synonyms
PRINCIPLES OF BIOASSAY
INDICATIONS OF BIOASSAY
TYPES OF BIOASSAY
USES OF BIOASSAY
DRAWBACKS
BIOASSAY IN HUMANS
3. What is ASSAY
Amount or activity of an active principle in unit
quantity of preparation
Types:
3
Physico-chemical
assay
Biological
(Bioassay)
Immunological assay
Photometry
Chromatography
RIA
ELISA
4. Bioassay
Potency or concentration of an active
principle in unit quantity of
preparation
by measuring its biological response
on living tissues
Introduced by Paul Ehrlich - biostandardization of
Diphtheria antitoxin
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5. Synonyms
5
Biological
assay
Bio
metrics
Biological
standadization
Bio-standadizatio
n
6. Principles of bioassay
To compare the test substance with the
International Standard preparation of the same
To find out how much test substance is required to
produce the same biological effect, as produced by
the standard
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7. Contd..
Activity assayed should be the activity of interest
Standard & test sample - similar pharmacological
effects & mode of action
Both should be compared for their established
pharmacological effect using specified technique
Ex: *Ach – contractile response on frog rectus
*Histamine – contractile response on guinea
pig ileum
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8. Contd..
Problem of biological variation must be minimized
Experimental conditions - kept constant
Animals - same species, sex and weight
Number of animals - large enough to minimize
error (individual variation)
Isolated preparations - sensitive
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9. Indications of bioassay
No chemical method has been developed
Chemical assay is too complex /not sensitive enough to
measure (ex: insulin, Ach)
To measure the pharmacological activity of new or
chemically undefined substances
For biological standardization of drugs obtained from
natural sources as these cannot be obtained in pure
form. Eg: Oxytocin,Vasopressin,Insulin,Heparin..
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10. Contd…
To compare the strength of a drug obtained
from various sources due to different
compositions (Eg:Cardiac glycosides)
Chemicals with similar structure, but different
biological activity
Chemical structure of the active principle is
unknown
Chemical structure known; cannot be actively
purified. Eg: Peptide hormones
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11. Characteristics of a good assay
method
Sensitivity
Specificity
Repeatability
Reproducibility
Precision
Accuracy
Stability – tissue has to stay “bioassay-fit
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12. Bioassay can be performed on
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• Intact
Invivo animals
• Isolated
tissues
• Specific cells
• Organisms
Invitro
14. ISOLATED TISSUE
Acetyl Choline – Frog Rectus Abdominus muscle
Histamine – Guinea Pig ileum
Adrenaline – Rat uterus
Oxytocin – Rat uterus oestrogen primed
DISPERSED CELLS
• Plasma LH estimation by stimulation of
testosterone synthesis - on isolated Leydig
cells
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15. Types of Bioassay
QUANTAL ASSAY
GRADED ASSAY
INDIRECT ASSAY
DIRECT ASSAY
16. Quantal assay
Quantal response - the response is in the
form of "all or none", i.e. either no
response or maximum response
Drugs producing quantal effect can be
bioassayed by end point method
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17. The threshold dose producing a predetermined
effect is measured
Comparison between the results of standard and
the test
E.g: Bioassay of digitalis in cats,Insulin induced
hypoglycemic convulsions in rat
Threshold dose of the Std
X Conc of Std
Threshold dose of the Test
Conc of Unknown =
18. Graded assay
Graded response - response is proportional to the dose
and response may lie between no response and the
maximum response.
Types:
• Bracketing /direct matching
• Interpolation
• Multiple point assays
Three point assay
Four point assay
Six point assay
• Cumulative dose response 19
22. Bracketing or Direct Matching
A constant dose of the standard is bracketed by varying
dose of test sample
until an exact matching between the response of std &
that of the sample is achieved
Strength of unknowm/test drug can be found by simple
interpolation of bracketed response.
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25. ADVANTAGES
Simple & Faster
Amount of test drug available is small
Does not involve complicated
calculations
Does not depend on DRC
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26. Disadvantages
less accurate,time consuming, troublesome
cannot get exact match of response
quantitative difference b/w test & std not
obtained
27. Interpolation assay
A log dose-response curve is plotted with the
standard on a simple graph paper or Semi-log
paper
The concentration of the test is then read from the
graph
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30. Advantages
Sensitivity of tissue is 1st determined by prior
plotting of a conc-response curve with known
agonist
Dose can be plotted even if it varies over thousand
fold range
Error is normally distributed
31. Disadvantages
Sensitivity of tissue changes with time
Timing of doses not taken into account
Variation in mode of application of drugs
32. Multiple point assays
Responses are repeated several times and the mean of
each is taken
Chances of error are minimized
3 point method - 2 doses of std+1 dose of test
4 point method - 2 doses of std+2 doses of test
6 point method - 3 doses of std+3 doses of test
Latin square method of randomization to avoid any
bias
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33. 34
t s1 s2
s1 s2 t
s2 t s1
3 - point assay
3 cycles
36. CALCULATION
• Mean responses of these 3 sets plotted
• Log potency ratio (M) =
(T-S1÷ S2-S1)× log d
where, d – dose ratio = s2/s1
• Strength of unknown =
s1/t × antilog of M
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40. Calculation
• Mean responses of 4 sets plotted
• Log potency ratio (M)
(T2-S2)+(T1-S1) × Log d
(S2-S1)+(T2-T1)
where, d-dose ratio = s2/s1
• Strength of unknown =
s1/t1 × antilog of M
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41. Six point assay
3+3 dose assay
3 conc each of std & test drug are used
6 sets of experiments using 6 doses in each set
More time consuming,lesser in use
Reliability is excellent
42. Cumulative Dose Response Curve
Increase conc of drug in bath fluid step by step
without washing out the preceeding doses
Continue till supramaximal effect is seen
Dose response curve is plotted
44. Uses of Bioassay
to measure the pharmacological activity of new/
chemically undefined substances
to investigate the function of endogenous
mediators
to measure drug toxicity and unwanted effects
to measure the conc of drugs and other active
substances in the blood or other body fluids
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45. Determination of potency, ED50/LD50 of
drugs
New drug development
Measure clinical effectiveness
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46. Drawbacks
• Biological variation
• Troublesome
• Time consuming
• Expensive
• Less accurate than physico-chemical
methods
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47. HUMAN TISSUE BIOASSAY
• Veins [surgery on varicose veins]
• Tissues like larger blood vessels obtained during
amputation
• Organs removed during transplantation/ tumor
surgeries/procedures requiring resection
• Tissues collected Postmortem
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48. Conclusion
Successful tool in estimation & discovery of biologically
active substances
Sensitivity & Specificity – important tool in pharmacology
Editor's Notes
The standards are internationally accepted samples of drugs maintained and recommended by the Expert Committee of the Biological Standardization of W.H.O.
In India, standard drugs are maintained in Government institutions like Central Drug Research Institute, Lucknow, Central Drug Laboratory, Calcutta, etc
Repeatability: Same observation using same
instruments and operators, and over short time periods
Reproducibility: Same observation using different instruments and operators, and over longer time periods
Estrogens – Ovariectamised Female Rat / vaginal cornification
. bioassay of digitalis in cats - Here the cat is anaesthetized with chloralose and its blood pressure is recorded. The drug is slowly infused into the animal and the moment the heart stops beating and blood pressure falls to zero, the volume of fluid infused is noted down. Two series of such experiments-one using standard digitalis and the other using test preparation of digitalis is done and then potency is calculated
Initial portion of curve is so steep that it is impossible to guage the magnitude of response corresponding to small increase in dose
When the response reaches max, large increase in dose required to produce small change in response
In arithmetic scale, it is difficult to display wide dose range
Biological standardization – comparison & adjustment of strength of the sample with that of std under controlled conditions
Limitation of animal tissues:
Species variable-cannot predict actual outcome in relation to the human