learning objectives: What is assay, definition of bioassay, its advantages & disadvantages ,principles ,types,examples and current status of bioassay.

1. Bioassays
Dr Uma Advani
Assistant professor pharmacology
SMS Medical College, Jaipur

2. Learning objectives
• What is assay ?Types of assays
• What is bioassay
• Indications of bioassay
• Advantages & Disadvantages of bioassay
• Principles of bioassay
• Types of bioassay
• Examples of bioassay
• Current status of bioassay
• Summary

3. What is assay?
• An assay is a procedure in molecular biology
for testing and/or measuring the activity of a
drug or biochemical in an organism or organic
sample

4. Types of Assays
• Chemical Assays:
• Spectrophotometry
• Spectrofluorimetry
• Chromatography
• Immunoassays
• Biological assays

5. What is bioassay : Bioassay
–Estimation of the conc. / potency of a
substance by measuring its biological
response in living systems
–i.e. Observation of pharmacological
effects on
• Living tissues or cells
• Microorganisms
• Intact animals

6. Indications for Bioassay
• Active principle of drug is unknown
• Active principle cannot be isolated, e.g.
insulin, posterior pituitary extract etc.
• Chemical method is either
– Not available
– If available, too complex,
– Insensitive to low doses e.g. Histamine can
be bioassayed in microgram conc.

7. …Contd.
• Unknown Chemical composition, e.g. long
acting thyroid stimulator.
• Chemical composition of drug variable but
has same pharmacological action e.g.
Cardiac glycosides isolated from different
sources, Catecholamines etc.

8. Applications of bioassay
1. Standardization of drug
2. Estimation of biologically active substances
like acetylcholine, Adr, NAdr in body fluid
3. Screening of new compounds for biological
activity
4. Diagnosis and research
5. Estimation of LD50 and ED50

9. Advantages of bioassay
1. Can detect very small concentration of the
substance
2. More reliable than chemical method as it is
done on a living organism/ tissue.

10. Disadvantages of bioassay
1. Less accurate
2. More laborious
3. Expensive

11. Principles of Bioassay
• All the bioassay must be comparative
against a standard drug or preparation
• The standard and the new drug should be as
far as possible identical to each other
• The degree of pharmacological response
produced should be reproducible under
identical conditions
– i.e. Adr. shows same rise in BP in the same
species under identical conditions: wt, age, sex,
strain / breed etc

12. …Contd.
• 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
• The method should be able to estimate the
error due to biological variations
• Bioassay might measure a different aspect of
the same substance compared to chemical
assay
• i.e. Testosterone & metabolites

13. Types of bioassay
Bioassay
Quantal Graded

14. Methods of bioassay in agonist
For
agonist
Quantal
End point
Graded
Matching Graphical

15. Methods for antagonists
• Commonly used method is simple graphical method
• Two responses of the same dose of agonist (sub
maximal giving approximately 80% of the maximum
response) are taken
• The minimum dose of standard antagonist is added in
the bath and then the response of the same dose of
agonist is taken in presence of antagonist
• The higher dose of standard antagonist is added and
responses are taken as before
• The percentage inhibition is calculated, plotted against
log dose of antagonist and the concentration of
unknown is determined

16. Methods for bioassays
Quantal
Direct
endpoint
Percentage
of positive
effect
Graded
Matching
assays
Multiple
Point Assays
Cross over
assays

17. Methods for bioassay
• Quantal Assays [ Direct endpoint ]
– Elicits an ‘All or None’ response in different animals
– The threshold dose producing a positive effect is
measured on each animal and the comparison
between the average result of two group of animals
is done
– Eg bioassay of digitalis on cat
• Drug is infused till the heart stops beating & BP falls to
zero
• The volume of infused is recorded
• Two series are done one with standard dose & other with
test dose
• Conc. of the unknown= (threshold dose of the standard/
threshold dose of the test) X conc. of standard

18. Contd…
– e.g.
–Calculation of LD50 in mice or rats
–Digitalis induced cardiac arrest in guinea
pigs and cat
–Hypoglycemic convulsions in mice.
–Digitalis induced head drop in rabbits

19. Contd…
Percentage of positive effect measured
• A quantal response is obtained & the percentage of the
positive effects at each dose is computed.
• Now the unknown is compared to the standard with
respect to potency in causing the quantal effects
• Eg LD50 estimation in toxicity testing
• The drug/ poison in different doses is injected into
animals and percentage of mortality is determined
• Next the percentage of mortality is plotted against the
dose and a curve is obtained and the dose causing 50%
mortality is thus obtained
• Other examples include assays of insulin

20. …Contd.
• Graded Response Assays [mostly on tissues]
• Graded responses to varying doses
• Unknown dose response measured on same
tissue

21. Methods of Bioassay
Cross over test
• The effect of the drug is measured more than once in
the same animal
• i.e on day 1, 1st set receives standard dose while the
second set receives unknown
• Now on another day, 1st set receives the unknown
while the first set receives the standard
• The average effect for each preparation for both the
stages is calculated & the results are interpreted by
drawing a log DRC
• Eg. Assays of insulin on rabbit
• It increases the accuracy

22. …Contd.
Matching / Bracketing: Const dose
bracketed with varying doses of standard till
exact match is obtained
• The conc. of unknown= (dose of standard/ dose of the
test) X conc. of the standard
• Used when test sample is too small
• i.e. Histamine on guinea pig ileum, Ach on frog
rectus, Posterior pituitary on rat uterus

23. Matching method

24. Contd…
• Disadvantages
– Inaccurate & margin of error difficult to estimate
– Match is purely subjective

25. …Contd.
–Multiple Point Assays
–3 point assay
–4 point assay
–6 point assay
–8 point assay

26. 3 point assay
[2+1 dose assay]
• Fast & convenient
• Procedure [e.g. Ach bioassay]
– Log dose response [LDR] curve plotted with
varying conc of std Ach.
– Select two std doses s1& s2 [ in 1:2 dose ratio]
from linear part of LDR
– Choose a test dose T between S1 & S2

27. …Contd.
– Record 4 sets data [Latin square:
Randomisation reduces error] as follows
• s1 s2 t
• t s1 s2
• s2 t s1
• s1 s2 t
– Plot mean of S1, S2 and T against dose. Calculate
– Log Potency ratio [ M ] =
[ (T –S1) / (S2-S1) ] X log d
– d = s2/s1
– Now calculate the conc of unknown as UK= s1/t X
potency ratioX conc of unknown
– Error is calculated as error= calculate- actual/actual X
100

28. 3 point assay

29. 4 point assay
[2 +2 dose assay]
• Procedure [Eg Ach bioassay]
– Log dose response [LDR] curve plotted with
varying conc. of std. Ach
– Select two std. doses s1& s2 from linear part
of LDR
– Choose two test doses t1 & t2
– Also s2/s1 = t2/t1 = 2

30. …Contd.
– Record 4 data sets with Randomisation
• s1 s2 t1 t2
• s2 t1 t2 s1
• t1 t2 s1 s2
• t2 s1 s2 t1
– Plot mean of S1, S2 and T1, T2 against dose.
Calculate
– Log Potency ratio [M] = [ (T1 –S1 + T2 –S2) / (S2-S1 +
T2-T1) ] X log d [d = dose ratio]
– d= s2/s1
– Now calculate the concentration of unknown(
unknown/ known = potency ratio
– Error is calculated as error= calculate- actual/actual X
100

31. Label
• s1= concentration of the standard
• s2= concentration of 2nd standard dose
• S1 = response due to s1
• S2 = response due to s2
• t1= concentration of 1st test dose
• t2= concentration of 2nd test dose
• T1= response due to t1
• T2= response due to t2

32. Contd…
• Microbiological assays
• Used for assaying antibiotics
• The test sample is compared with standard preparation
for its inhibitory effect on particular organisms

33. Drugs Preparation Activity assessed
Acetylcholine Isolated rectum
Rectus of frog
Rat ileum
Isolated mouse heart
Leech dorsal muscle
Rat/cat BP
Contractile effect
Contractile effect
Contractile effect
Inhibition of contractions
Contractile effect
Fall in BP
Histamine Isolated, atropinized
terminal ileum of guinea
pig
Anaesthetized and
atropinized cat
Contractile effect
Fall in blood pressure
Adrenaline BP of spinal cat
Isolated rabbit duodenum
Isolated rat uterus
Isolated caecum of fowl
Rise in BP
Inhibition of tone
Inhibition of tone
Inhibition of tone
Bioassay of Some Important Drugs

34. Contd…
Drugs Preparation Activity assesed
Digitalis Cat blood pressure
Guinea pig blood pressure
Pigeon
Fall in BP and death
Stoppage of heart & death
Emesis
5 HT Isolated atropinized rat
uterus,
Isolated terminal colon of
rat
Isolated fundus strip
of rat stomach
Perfused rabbit ear
Contractile effect
Contractile effect
Contractile effect
Constriction of blood
vessels
Heparin Whole blood of
ox with thrombokinase
extract and acetone
dried ox brain
Prolongation of blood
clotting time
Noradrenaline BP of pithed cat Rise in BP

35. Contd…
Drugs Preparation Activity assessed
Insulin Rabbit
Mice
Isolated rat diaphragm
Rat's epididymal fat
Lowering of blood-sugar
Convulsions and/or death
due to hypoglycaemia
Increase in glycogen
content
Increased metabolism
of glucose, indicated
by increased in CO2
production
Oxytocin Adult cockerel
Isolated rat uterus
Rabbits (female)
Vasodepressor activity.
Contractile effect
Ejection of milk from
mammary duct

36. Current status of bioassay:
• If one review the emphasis of bioassay in
pharmacopeias published before 1980 as
compared to those published recently, it will be
clear that
1. There are very few drugs which are now
recommended to be assayed by biological methods.
2. Most of the drugs which were assayed by biological
methods are now being recommended to be
assayed by chemical methods
3. Newer drugs have been included for which bioassay
recommended

37. Contd…
• Recently newer techniques have come up
which have replaced the bioassay techniques
• Eg chromatography for estradiol and
penicilline & HPLC for insulin

38. To summarize
• Bioassay are defined as estimation of the
conc. / potency of a substance by measuring
its biological response in living systems
• They are indicated when the active principle
of the drug is unknown, chemical method is
not available or is too complex or when the
composition of the drug is variable
• They have been applied for variety of purpose
like standardization of the drug or for research

39. Contd…
• Biological assay are of two types viz: Quantal and
the Graded type
• Quantal( all or none response) can be measured
using end point method or % of positive effects
• Graded can be measured using matching analysis
or multiple point assays
• Now a days with the advent of newer and more
accurate techniques the use of bioassays is on a
decline

40. Thank you