BIOASSAY &

BIOSTANDARDISATION
Dr. Anoosha Bhandarkar
Post Graduate
Department of Pharmacology
SDM Medical College
21-07-2011
1
OVERVIEW
Experimental Pharmacology
Assay
Bio assay
When bioassay?
Applications
Principles
Types of Bio assay
Requirements
Merits & demerits
Different tissues used
Human tissue bioassay

2
EXPERIMENTAL PHARMACOLOGY
Aims:
To find out therapeutic agents suitable for human
use
To study MOA & site of action
To study toxicity of drugs acute , subacute, chronic
Types : qualitative ( analyse activity) & quantitative
( assay activity)
3
BIOSTANDARDISATION
Introduced in 1920s by Paul Ehrlich : bio-standardization of
Diphtheria antitoxin by his side-chain theory of immunity.

Defn: Comparison and adjustment of the strength of the
sample with that of the standard under controlled conditions
Necessary to regulate the doses of crude extract.

4
ASSAY
Definition:
Quantitative estimation of drugs or of their active
constituents
Types
Physico-Chemical Assays: eg. Salicylates, Sulfonamides etc..
M/commonly used procedure . Eg. Spectrophotometry,
Chromatographic & mass spectrometric techniques
Immuno - Assays : RIA , immunoradiometric assays for
protein hormones & drugs
Biological Assays (Bioassay)
Microbiological assay
Radio-receptor assays: principles of Bioassay + Radioimmuno
assay

5
SELECTION OF METHOD:-


PHYSICAL PROPERTY
LIKE COLOUR ,
FLOURESCENCE
PHYSICAL

PHYSICAL PROPERTY
& CHEMICAL
REACTION
PHYSIO-CHEMICAL

BIOLOGICAL
PROPERTY USED TO
ESTIMATE ACTIVITY
BIOLOGICAL
BIOASSAY
Estimation of concentration or potency of a substance/drug
from the magnitude of its main biological response.
Main pharmacological effect of the drug under test is compared
with that of standard drug & their potency ratios are compared
quantitatively
Classified : Qualitative & Quantitative
7
.

Qualitative bioassays : used for assessing the physical
effects of a substance that may not be quantified,
- abnormal development or deformity.
Eg. Arnold Adolph Bertholds experiment
on castrated chickens



Quantitative : estimation of the concentration or potency
of a substance by measurement of the biological
response that it produces.
Analyzed using the methods of biostatistics.
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Whole animal
Isolated
tissue
Cells for in
vitro study
Micro-
organisms

Observation of pharmacological effects on :
Isolated tissues or cells for invitro study
Micro-organisms
Whole animals singly/groups
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10
TISSUE

1. Whole
animal
SUBSTANCE

a. Vasopressin



b. Estrogens



c. Vit D


d. Insulin


e. d-tubocurarine
RESPONSE ELICITED

- Anaesthetised rat for rise in
B.P
- Hydrated rat for reduction in
U/O


- ovariectemised female rat for
vaginal cornification


- Rat, alleviation of the rachitic
state


-- mice, hypoglycemic
convulsions or death

-- Rabbit, head drop d/t paralytic
relaxation of skeletal muscles
11

2. Isolated
tissue

a. Ach

b,
Histamine

c. Adr

d. Oxytocin


e. 5HT

-Frog, rectus muscle
contraction

- Guinea pig ileum, contraction

-Rat uterus in diestrus,
relaxation

- Rat uterus estrogen primed,
contraction

- Gastric fundus contraction

TISSUE


cells dispersed in a suitable
medium

RESPONSE ELICITED

IN VITRO measurement of
concentration of Plasma LH by
Levels of testosterone synthesis by
isolated Leydig cells of testes


Micro organisms


Vitamin B12: Growth of Euglena
gracilis
12
INDICATIONS FOR BIOASSAY
Chemical composition is not known but has a specific
biological action. Eg. LATS

Chemical assay method is too complex/ insensitive.
Eg. Adr, His can be bioassayed in micrograms

Drugs differ in composition having same pharmacological
action. Eg. Digitalis glycosides

When active principle is unknown/ cannot be easily isolated.
Eg. Peptide hormones- insulin, GH


13
BIOASSAY GENERALLY EMPLOYED IN..
Estimate concentration/potency of known active principle in
tissue extract.e.g..insulin; body fluids Serotonin
Estimate pharmacological activity of new/ chemically
undefined substance agonists / antagonists on a tissue
Comparing competitive v/s non-competitive antagonist
(antagonists assay)
Measure drug ED
50,
LD
50
& adverse effects
Investigate function of endogenous mediators- Tyramine
Subtype of receptors & Tissue selectivity of drug
Diagnostics, toxicity studies , research.


IT IS ONLY METHOD UNDER FOLLOWING
CONDITIONS..
If active principle of drug is unknown. e.g. insulin.
Chemical method not available.
Chemical composition not known.
Quantity of the sample is too small. Eg. micrograms
Purification for chemical assay not possible.
As quantitative part of screening procedure.
Investigate function of endogenous mediators- PGs, EDRF,
hypothalamic factors
Measure drug toxicity & adverse effects
STANDARD CHEMICALS
Representative of a substance serves as basis for comparative
measurement of activity.
Internationally agreed upon standards are necessary compare
potency
A stable standard solution has to be employed for comparison
Standards for sera held at State Serum Institute, Copenhagen
- National Inst.for Med Research(UK)
16
In India Standard chemicals maintained & distributed by:
Central Drug Research Laboratory, Kolkata.
Central Research Institute, Kasauli
PRINCIPLES OF BIOASSAY
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Comparison of the main pharmacological response of the
unknown preparation with that of the standard
When pure substance unavailable standardised prep. of
hormones/ natural products used

Ref standard & Test sample should as far as possible identical,
viz. pharmacological effects, mode of action, LDR run parallel &
potency ratios conveniently compared

Specific effect produced by the active principle must be the same
in all animal species

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Certain quantity of drug produces same degree of response in
same animal or animals of same species under identical
conditions. Eg. Adr same degree of rise in BP

Compared for their established pharmacological effect using a
specified pharmacological technique . Eg. ACh on frog rectus &
His on guinea pig ileum/tracheal ring chain prep.

Assayed activity should be the activity of interest
Method should minimise /estimate as far as possible the error
due to biological variation.

REQUIREMENTS ..
High sensitivity
Specificity. Eg. Guinea pig ileum atropinised for the
bioassay of histamine
Reproducibility- response with the dose shoild remain the
same
Accuracy
Stability tissue has to stay bioassay-fit
Easy availability of animals
19
TYPES OF BIOASSAYS
Indirect assays : potency of sample estimated by
comparing LDR curve of sample with the similar curve of the
standard
Eg. Bioassay of crude ergot preps.
Ergot preparations injected in the whiteleghorn cock
vasoconstriction bluish discoloration of comb

Direct assays : dose of the sample required to elicit a
particular pharmacological effect (ED50 or LD50) is
measured . Eg. Death in guinea pigs
20
DIRECT ASSAYS
1. Quantal assay: direct end-point
assay
The dose of the std &
that of the unknown producing a
Predetermined all or none response is
measured & potency ratios compared.
Dose is known as tolerance/threshold
dose
Ratio of TD50 sample/ standard relative
potency of sample
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22
LDR curves plotted
b/w cumulative % subjects showing cardiac arrest
& respective threshold doses
The volume of fluid passed into the vein noted for
each animal THRESHOLD DOSE
Two series of experiments done : std. & test
solution using atleast 10 cats/ series
BIOASSAY OF DIGITALIS
extract containing digitalis( std/ unknown) infused into
vein of the anaesthetised cats at 1ml/min till heart stops &
BP falls to zero level
TD50 (threshold dose producing cardiac arrest in 50% of the
subjects) is then calculated for standard (log x) & unknown (log
x1)

Toxicity(LD50) & potency(ED50) ratios are calculated & compared

Strength of the unknown calculated
Eg.
D-tubocurarine induced head drop in rabbits
Insulin induced hypoglycemic convulsions in mice
Calculation of LD50 of drugs in mice or rats

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2. Graded Response Assays [mostly on tissues]
Graded responses to varying doses of a drug
Proportionate increase in responses with increase in dose
Effect of both , the standard drug & the Unknown measured
repeatedly on the same tissue
Eg. Bioassay of histamine on guinea pig ileum
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BASIC REQUIREMENTS FOR BIOASSAY
Instrumentation
Physiological salt solution(PSS)
Procedures & drugs to render the animals
unconscious
Tissue isolated / whole

25
INSTRUMENTATION
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Rudolph Magnus-1904
APPARATUS
Outer water bath of perspex/glass
Inner organ bath {single or multiple}- glass. 15-
100ml
Tissue holder cum O
2
tube
Glass coil connected to organ bath, mariotts bottle
Thermostat
Electrical stirrer
Writing lever

Platinum electrodes embedded in plastic for
innervation of nerves
Recording drum, Adjustable clamps, holders, grips,
X blocks, dissection instruments etc.
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Frontal lever, Gimbal lever Sprung
lever
SALT SOLUTION
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Frog ringer: Frog heart & tissue, Ringers solution: Mammalian isolated heart & other
tissues
Tyrodes: Mammalian smooth muscle, Krebs: Mammalian isolated organ specially for
nerve responses, DeJalon : No Mg2+/PO4+ ions
PROCEDURES TO RENDER ANIMALS UNCONSCIOUS
Mice, rats, guinea pigs & rabbits : stunning ( crushing the skull
) followed by cutting the throat for bleeding
Frogs : - Chilling to 4C until state of immobility reached
- Pithing single & double (unless & otherwise)
Lab. anaesthetics used:
Barbiturates mostly used.
Eg. Pentobarbitone sodium(NEMBUTAL), phenobarbitone sodium
Produces anesthesia in dose of 35-45mg/kg. i.p./i.v.Duration 45-60 min.
Others : chloralose (80-100mg/kg,ip or iv)& urethane (1-1.5g/kg,ip or
iv), paraldehyde (2-2.5mg/kg i.m / i.v), 20% MgSO4 (5ml/kg i.v )

29
Tissue Use-Assay Cycle Remarks
Frog Rectus
abdominus
Frog Ringer

1. NM blockers
2. N
m
receptor agonists
3. Analyzing AntiChE
activity/Susceptibility to
ChE
Drug 90s
Rest- 4-4.5
min
30 min relaxation
prior to start
Sturdy, slow
contracting
Dorsal
muscle of
Leech
Frog Ringer
or Ringer
Locke soln-
5:7 dil
1. NM blockers
2. N
m
receptor agonists
3. Analyzing AntiChE
actvity/Susceptibility to
ChE
Drug 90s
Rest- 13.5
min
Delicate tissue
Most sensitive to Ach
after addition
physostimine
Guinea pig
ileum
Tyrode soln.

Agonist and Antagonist on
Muscarinic, Histaminic,
5HT, nicotinic, Bradykinin
receptor
Drug 30s
Rest- 2.5 min
Delicate tissue, Non-
specific, May show
spontaneous activity
Rat Phrenic
nerve
diaphragm
Krebs
solution

NM blockers
Local anaesthetics- Nerve
block

Drug -3 min,
Rest- 6 min
Care of the nerve
Drain by overflow

30

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Tissue Use-Assay Cycle Remarks
Rabbit
Jejunum
Tyrode
adrenergic agonist &
antagonist
Drug 30s
Rest- 150 s

Spontaneous pendular
movements inhibited
by adrenergic
agonist
Finkelman
Preparation:
Rabbit
jejunum with
mesentery
adrenergic antagonists
Adrenergic neurone blocking
agents
Nerve block local
anaesthetics

Slow rate
2-4/sec
Parasymp.
Fast rate
30-50/sec
Symp.
stimulation
Mesentery contains
sympathetic &
Parasympathetic
nerves

Rat Fundus
Krebs
solution
5HT agonist & antagonists

Drug 90s
Rest- 4.5 min

Relax for 30 min
before experiment

Rat Uterus
De Jalons
low Ca
++
Temp: 30
Oxytocin & its antagonist,
Ach,
adrenergic agonist and
antagonist
Drug 30 s
Rest- 2.5 min

Pre-treatment with
stilbesterol

Guinea pig-
Tracheal
chain
Krebs
solution
Histamine, ACh, 5HT,
adrn agonist & antagonist

Drug 5 min
Rest- 10 min

Special manner of
tying the ring

METHODS OF DOING GRADED BIO ASSAY
Matching bioassay
Bracketing assay
Interpolation method
Multiple point bioassay
Three point bioassay
Four point bioassay
Six point bioassay
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Not reliable
Not consider sensitivity change w.r.t time,
timing of doses, variations in methods of
application of drugs & Few dose analysis
MATCHING BIOASSAY
Employed for small sample size
1. Firstly responses of the test of a particular dose is taken
2. It is matched with the dose of the standard (whose strength
is known) by trial & error method
3. Done till a closed matching is observed.
4. Corresponding concentration calculated.
5. Potency ratio of the two can be approximately found &
strength of the unknown test solution can be calculated
6. Eg. histamine bioassay, posterior pituitary assay on the rat
uterus
33
go
BRACKETING METHOD
Used when test sample is too small
1. Response of test is bracketed b/w two responses
(greater & smaller) of standard substance.
2. Strength of unknown found by simple interpolation of
this bracketed response on the dose axis
Precision & reliability is poor.

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BRACKETING METHOD
35
back
MATCHING/ BRACKETING
Advantage:
Faster
Can be completed when amount of test drug available
is small
Does not involve complicated calculations
Disadvantage
Match is subjective
Exact match may not always be possible
No evidence of parallelism/ discrimination
Does not permit calculation of variation.
36
INTERPOLATION METHOD
1. Log dose response curve of std. is established
with atleast 4 submaximal concentrations
1. 2-3 responses of test is recorded.
2. Select dose responses that lie on LINEAR
PORTION of LDR curve of standard
Strength of unknown found by interpolation of these on dose axis &
taking antilog
Advantage:
Faster
Can be completed when amount of test drug available is small
Disadvantage:
No evidence of parallelism/discrimination
Does not permit calculation of variation/ precision
37
THREE POINT BIOASSAY [2+1 DOSE ASSAY]
Fast & convenient procedure
LDR curve plotted with varying conc. of std & test solution
2 standard, s1& s2 [1:2 dose ratio] from linear part of LDR & 1 test
response [between s1 & s2]are taken
Record 4 sets data
Latin square: Randomisation reduces error
Plot mean responses of S1, S2 and T against dose.
Calculate Log Potency ratio
M = [ (T S1) / (S2-S1) ] X log d [d = dose ratio]
Strength of unknown calculated as
Strength = s1/t (antilog of M)
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THREE POINT BIOASSAY [2+1 DOSE ASSAY]
39
CHART CALCULATION

Respons
e
Hts of
contract
n
1
(mm)

2

3

4

Mean
(mm)


Dose(ml)

Dose
ratio

S1

35

40

40

45

40

0.1


s2/s1
= 2

S2

75

80

80

85

80

0.2

T

55

60

60

65

60

0.25
40
Log potency ratio M = [ (T S1) / (S2-S1) ] X log d where [d = dose ratio=2]
M= 0.150; Strength of the unknown = s1/t * (antilog of M) = 0.5652 potency of std.
FOUR POINT BIOASSAY
Most common method used; 2 standards (s1,s2) & 2 test (t1,t2) responses
Two responses of standard & test should lie on linear portion of CRC in ratio of
1:2
4 sets of bioassay are done using Latin square design
Plot mean responses of s1, s2 & t1, t2 against dose
Horizontal separation: log potency ratio(M) of the conc. of T & S
M= {(T1-S1+T2-S2) / (S2-S1+T2-T1)} * log d. d = dose ratio
Strength of T = (s1/t1) * antilog of M
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FOUR POINT BIOASSAY

42
CHART CALCULATION

RESPONS
E
Hts of
contractio
n
1 (mm)

2

3

4

Mean
( mm )

Dose
Dose
ratio

S1


35

40

40

45

40

0.1

S2/S1

S2


75

80

80

85

80

0.2

t2/t1=2

T1

40

45

45

50

45

0.15

T2

80

85

85

90

85

0.3
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Log potency ratio M= {(T1-S1+T2-S2) / (S2-S1+T2-T1)} * log d d = dose
ratio = 2
M = 0.037; Strength of unknown soln T = (s1/t1) * antilog of M = 0.726 potency of std.
SIX POINT BIOASSAY (3 + 3 ASSAY)
Reliability is excellent
Time consuming
Three concentration of standard & test are used.
6 sets of experiments using 6 doses in each set
6*6 = 36 doses in latin square design

Recent applications:
- Microbiological assay of vit B12
- Analgesic assay of sublingual buprenorphine & i.m
morphine
- Diazymes cystatin-C assay emerging markerrenal
disease
- Comparitive assays of Erythropoietin standards


44
CUMULATIVE DOSE RESPONSE CURVE
45
C-DRC is obtained when the drug is added with increasing
concentration without washing the previous dose
Tissue sensitivity, tachyphylaxis, antagonist assays
R
e
s
p
o
n
s
e

MERITS DEMERITS
Biological products like toxin,anti
toxin,sera can be conveniently
assayed.
Key problem is variability in
response.
Always not reproducible
Measure minute (nano & Pico mole)
quantities of active substances.
Large number of animal to be used.
Expertise in experimental design,
execution of assay & analysis of
data required.
Can detect active substance without
prior extraction or other treatment.
Tachyphylactic responses of
substance being assayed.
More chemicals in single animal
tested
Expensive & time consuming.
Unwanted effects on other system
avoided

Time related changes in sensitivity of
test organ.
No neuronal response / reflex
mechanism
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Antagonists assay
Competitive antagonism
Isolated rabbit aortic strip
Cumulative DRC for NA before &
after Phenoxybenzamine
Max response can be obtained with
increase dose
Parallel shift of curve to right side with
antagonism
Non-Competitive antagonism
Isolated guinea pig ileum
Cumulative DRC of Histamine before
& after Phenoxybenzamine
Max response is not achieved even
with higher dose of Ag
Flattening of the curve
Physiological antagonism-
Cholinergic v/s adrenergic
Isolated rat colon
Contractile response to carbachol
inhibited by Adr
Non-Specific antagonism
Isolated rat colon
Contractile response to carbachol
inhibited by Papaverine
DRC- Dose response curve
NA Nor-Adrenaline
ADR- Adrenaline
Phenoxybenzamine- Non selective antagonist
Carbachol cholinergic agent
Papaverine- direct smooth muscle relaxant property
47
HUMAN TISSUE BIOASSAY
Limitation of animal tissues:
Species variable: cannot predict actual outcome in relation to human
Use of human tissue
use of cell lines with close precision to human
Human tissues that can be used
Veins [obtained from surgery on varicose veins]
Larger blood vessels obtained during amputation
Organs removed during transplantation/ tumor surgeries/ procedures
requiring resection/ aborted foetus
Tissues collected Post mortem
48
Interestingly, clinical trials for assessing drug
effects in humans involve similar principles
as bioassays in animals!!

Environmental bioassays for effluent toxicity
tests of sewage & industrial wastes is a must
for municipal sewage Rx plants at U.S
49
REFERENCES
Sharma & Sharma. Principles of Pharmacology. Revised ed.
2011
MN Ghosh. Fundamentals of experimental Pharmacology.4
th

ed. 2008
Pharmacology & pharmacotherapeutics by Satoskar &
Bhandarkar, revised 21
st
edition
Internet search
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