This slide id very good for students

1. BIOAS
S
AY

2. An assay is an investigative (analytic) procedure
pharmacology,
medicine,
biology, and molecular
assessing or
in laboratory
environmental
biology for
quantitatively
qualitatively
measuring the presence or
amount or the functional activity of a target
entity (the analyte) which
biochemical
can be
substance or
a drug or
organic sample

3. Chemical Assay
Immunoassay
Bioassay

4. Chemical Assay:
It is the study of the separation, identification, and
quantification of the chemical components of natural and
artificial materials.
Immunoassay:
A technique that makes use of the binding between an antigen
and its homologous antibody in order to identify and quantify the
specific antigen or antibody in a sample.

5.  Comparative assessment of relative potency of a test compound to a
standard compound on a living or biological tissue.
 Quantitative measurement of the amount of active principle or substance in
a pharmaceutical preparation or biological material using a suitable
biological system
Introduced by Paul Ehrlich - biostandardization of Diphtheria antitoxin

6. Bioassay Chemical Assay
Less Precise
More ti
B
m
ioassay
e consuming
More expensive
Active constituent &
structure not known.
More sensitive
More men power
Required
Difficult to handle
More Precise
Less time consuming
Less expensive
Active constituent &
structure fully
established.
Less sensitive
Less men power
required
Easy to handle

7. Biological
assay
Bio
metrics
Biological
standadizatio
n
Bio-
standadizati
on

8. 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
Activity assayed should be the activity of
interest

9. 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
abdominis muscle
*Histamine – contractile response on guinea
pig ileum

10. 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

11.  No chemical method has been developed
 Chemical assay is too complex /not sensitive enough to
measure (ex: insulin)
 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..

12.  To compare the strength of a drug obtained
from various sources due to different
compositions
 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

13. Sensitivity
Specificity
Repeatability
Reproducibility
Precision
Accuracy
Stability – tissue has to stay “bioassay-
fit

14. • Intact
animals
Invivo
• Isolated
tissues
Invitro

15. WHOLE ANIMALS
Nor Adrenaline – Cat
Cardiac Glycosides – Guinea Pig
Insulin – Mice
Estrogens – Ovariectamised Female Rat
ISOLATED TISSUE
Acetyl Choline – Frog Rectus Abdominus muscle
Histamine – Guinea Pig ileum
Adrenaline – Rat uterus
Oxytocin – Rat uterus oestrogen primed

16. Qualitative bioassay
Is used for assessing the physical effects of a substance that
may not be quantified, such as abnormal development or
deformity.
Eg: Arnold Adolph Berthold's famous experiment on castrated
chickens. This analysis found that by removing the testes of a
chicken, it would not develop into a rooster because the
endocrine signals necessary for this process were not available.
Quantitative bioassays
involve estimation of concentration/potency of a substance by
measurement of the biological response it produces. These
bioassays are typically analyzed using the methods of
biostatistics

17. 1.Direct end point assay (DEPA)
2.Quantal assay (all or none assay)
3.Graded assay :
a) Bracketting assay
b) Matching assay
c) Interpolation assay
d) Multiple point assay ( 3-point, 4point, 6 -
point, 8-point).

18. Principle: is to measure direct response of dose of standard and
test preparation. The threshold dose required for response is
determined for each experimental unit.
Ratio between these doses estimates the potency of the test
preparation relative to the standard.
Concentration of test= TDS/TDT×CSD
Where, TDS= Threshold dose of standard
TDT= Threshold dose of test
CSD= Concentration of standard drug
Threshold dose of standard = Total period of infusion × rate
of drug administration

19. Advantage:
Drug effects appear rapidly and are easily recognised
Drug effect is directly proportional to drug dose
Rapid end-point detection.
Disadvantages:
Only toxicity study or high dose study is possible
Dose ranging study cannot be done

20.  Quantal response – the unknown is compared with the standard with
respect to potency which produces the quantal affect, i.e change is easily
recognised sign or often death.
 In a quantal assay there is use of dose response relationship.
 quantal response to a drug is obtained and percentage of positive
response at each dose is calculated.
 The response in quantal assay is varying, i.e some responses are
irreversible and hence animal used is once and some responses have no
permanent effect and animal can be used in next experiment
 e.g –
 Determination of LD50
 Hypoglycemic convulsion in mice in the assay of insulin
20

21.  Graded response - In these assays, as the dose
increases there is an equivalent rise in response.
The potency of a test agonist is determined by
comparing its mean response to standard mean
response.
 This process is also known as “analytical dilution
assay”
 GRA is the simplest way of determining potency of a
test drug because it does not require statistical
analysis

22. standard
Test/unknow
n

23. DRC & Log DRC
30%
70%
Sigmoid curve
Wide range of doses
can plot
 Rectangular hyperbola
• Potency
• Efficacy
• Slope of curve

25. Used when test sample is too small.
Comparison of potency between unknown and standard drug is done
by trial and error method.
Response is matched at only one dose
Does not need dose respone curve of test compound
It require most sensitive tissue
Concentration of test= (Dose of standard/ Dose of test)× conc. Of standard
Disadvantage:
Experimental error is not excluded out
There is no sign of parallelism as it lack dose response relationship.

26. Used when test sample is too small.
Single or few responses is taken by using any test drug
concentration.
This response is bracketed between two responses one
higher and one lower of the standard drug.
The strength of the unknown can be found by simple
interpolation of this bracketted response on dose axis

28. less accurate,time consuming, troublesome
cannot get exact match of response
quantitative difference b/w test & standard not
obtained

29. standard
Test/unknow
n

30.  A log dose-response curve is plotted with the standard,
 Single or few responses of test drug are plotted.
 The dose of test drug which comes at the linear log dose-
response is interpolated from the dose respone plot

31. 100
50
0 x x1
standard
%
R
E
S
P
O
N
S
E
LOG DOSE

32. 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

33. Sensitivity of tissue changes with time
Timing of doses not taken into account
Variation in mode of application of drugs

34. 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
35

35. 36
t s1
s2
s1 s2 t
s2 t
s1
3
cycles

36. 37
%
R
E
S
P
O
N
S
E
T
S2
s1 t s2
LOG DOSE
S1

37. s1 s2 t
t s1 s2
s2 s1 t
t s2 s1

38. • 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
S1, S2- length of standard dose response selected between
25-75%
T-length of test dose response selected in between of
two standard responses
s2/s1 -standard drug dose which came in contact with tissueand
given the response S1, S2. respectively
t = Test drug dose which came in contact with tissu and given the
response T
39

40. 41
4 - POINT ASSAY

41. 42
%
R
E
S
P
O
N
S
E
T1
S2
s1 s2 t1
LOG DOSE
S1
T2
t2

42. s1 s2 t1 t2
s2 t1 t2 s1
t1 t2 s1 s2
t2 s1 s2 t1

43. • 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
44

45. 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

46. 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
47

47. Determination of potency, ED50/LD50 of
drugs
New drug development
Measure clinical effectiveness
48

48. • Biological variation
• Troublesome
• Time consuming
• Expensive
• Less accurate than physico-chemical
methods
49

49. Successful tool in estimation & discovery of
biologically active substances
Sensitivity & Specificity – important tool in
pharmacology