Bioassay: A method for estimate the unknown drug concentration in given solution

1. Principles of
Bioassay
Presenter:-
Dr Arun Singh
Senior Resident
Department of Pharmacology
SMS Medical College, Jaipur

2. Learning Objectives
History Definition Introduction
Methods of
Estimation of
Concentration of
Unknown Drug
Principles of
Bioassay
Applications of
Bioassay
Classification of
Bioassay
Three Point Assay
Four Point Assay

3. History
Bioassay was started in late 18th century, when
standardization of Diphtheria antitoxin was done by Paul
Ehrlich.
Thereafter, it was a common practice to standardize any
substance through biological assay.
Bioassay is a most important basic step towards the drug
discovery and can determine the effect of any natural source
of unknown substances without affecting complete system.
So, it is the method for the estimation of the potency of a
material on living system.

4. Definition
Bioassay comprises of “bio” means living
material and “assay” means assessment at
laboratory, i.e., assessment of unknown
substance on any living tissue.
Hence, bioassay is defined as comparative
assessment of relative potency of a test
compound (T) to a standard compound (S)
on any living animal or biological tissue.
This procedure is for determining the
quantitative relationship between the
concentrations (dose) and magnitude of
response.

5. Introduction
Bioassay is an analytical method to determine the
concentration or potency of a substance by its effect
on living animals or plants (in vivo), or on living cells or
tissues(in vitro).
A bioassay experiment may be quantal or quantitative,
direct or indirect.
Qualitative bioassays are used for assessing the physical
effects of a substance that may not be quantified, such
as abnormal development or deformity, whereas
quantitative bioassay assessed at the laboratory level
and the concentration or drug dose can be evaluated.

6. Methods for Estimation of Concentration of Unknown Drug
• The quantitative estimation of drugs or of their active constituents can be done
by:
(1) Biological methods (bioassay)
(2) Physicochemical methods (e.g., by chromatographic, spectrophotometric,
fluorometric and mass spectrometric techniques)
(3) Radio-immunological methods
(4) Microbiological methods

7. Objectives
a) To measure the pharmacological activity of new or chemically undefined
substances
b) To measure the concentration of known substances (e.g., acetylcholine,
histamine, serotonin or catecholamine) in a tissue extract or body fluids
c) To measure ED50 or LD50 of a drug
d) For biological standardization of drugs from natural sources which cannot be
obtained in a chemically pure form, e.g., vasopressin, oxytocin, insulin and heparin

8. Principles of Bioassay
(I) The bioassay usually involves the comparison of the
main pharmacological response of the unknown
preparation with that of the standard.
• The best kind of standard, of course, is the pure
substance; but at times it is often necessary to utilize
the standardized preparations of hormones or natural
products against which the unknown sample can be
calibrated, even though the standard preparation
used is not chemically pure.

9. II) The reference standard and the test sample should have similar
pharmacological effects and should have the same mode of action, so
that their log dose-response (LDR) curves run parallel, and their
potency ratios can be conveniently compared.

10. III) The test solution and the reference standard should be compared
for their established pharmacological effect using a specified
pharmacological technique, e.g., acetylcholine and histamine can be
compared with their respective test samples for their contractile
responses on frog rectus abdominus muscle or isolated guinea pig
ileum (or tracheal ring chain preparation), respectively; adrenaline and
its related test sample for the rise in blood pressure in dogs or cats, and
so on.
• However, some exceptions can be made, e.g., insulin preparations should ideally be
assayed by estimating blood sugar levels, but, for the sake of convenience, the most
commonly used method is the comparison of hypoglycemic convulsions in mice.

11. IV) The methods selected should be reliable, sensitive,
reproducible, and should minimise errors due to
biological variations and methodology.
• Hence, the animals used should always be of the same species, sex
and weight and if an isolated preparation is used, it should be
sensitive.
• The number of animals should be large enough to permit statistical
analysis and the experimental conditions should always be kept
constant.

12. Application of Bioassay
• According to several pharmacopeias,
assay used for the drug may be
varied. For example:
1. Estimate potency of natural drug,
for which chemical method is not
known or established
2. Standardization of drugs of natural
origin (plant and animal origin) whose
structure or origin is unpredictable

13. 3. Screening of new compound for
biological activity
4. Estimation of biologically active
substance like histamine,
acetylcholine, 5-hydroxytryptamine,
adrenaline, bradykinin, substance P,
prostaglandins, etc.
5. Estimation of ED50/TD50 and
LD50.(Presently instead of LD50,
NOAEL or LD10 is preferred).

14. Classification of Bioassay
• Broadly bioassay is classified into three
groups namely:
1. Direct endpoint assay (DEPA)
2. Quantal assay (all or none assay)
3. Graded assay
a. Bracketing assay
b. Matching assay
c. Interpolation assay
d. Multiple point assay (3-point, 4-
point, 6-point and 8-point)

15. Direct End-point Assay
In a direct assay, the threshold dose
required for response is determined
for each experimental unit.
The principle of direct assay is to
measure direct response of dose of
standard and test preparation.
The ratio between these doses
estimates the potency of the test
preparation relative to the standard.

16. The response should be clear and easily recognized and the dose
given to the experimental animal should be in such a way that is
easily measured.
Thus, the observed data are dose units, e.g.: assay of digitalis in cat.
Unlike, the indirect assay the experimental unit receives one or more
specified doses of the preparation, and the observed data may be
either quantal or quantitative responses.

17. Depending on the experimental design, several dose levels of T
and S are given to the same or different experimental units and
the mean dose level is selected.
Mainly two experimental designs are followed, one is
crossover, and another is parallel group or completely
randomized design.
In the procedure, the test or standard preparation is infused at
a fixed rate into the circulation of animal until a direct effect is
observed in the animal.
For example: stopping of heart beating after the continuous
administration of digitalis at the constant rate in the assay of
digitalis in cats.

19. Advantages:
• Drug effects appear rapidly and are easily
recognized
• 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 Assay (All or None Assays)
• The unknown is compared with the
standard with respect to potency which
produces the quantal affect, i.e., changes is
easily recognized sign or often death.
• These responses are recorded; reason being
effect of some drug or stimulus to any
targeted system is not able to record
response quantitatively or the reaction of
subject is so minimal which cannot be
quantified or recorded.

21. • In a quantal assay there is use of dose response relationship,
however assay are more closely related to direct assay.
• As a procedure quantal response to a drug is obtained and
percentage of positive response at each dose is calculated.
• Most popular example in the drug discovery is determination of
LD50 and other is assay of insulin by mice convulsion method.

22. • The response in the quantal assay is varying, i.e. some responses are
irreversible and hence the animal used is once but some responses have no
permanent effect and can be used in a design of several test.

24. Graded Response Assay (GRA)
In this type of biological
assays, the extent of the
reaction is a function of
the dose of drug.
Ideally, the quantitative
relation between dosage
and response would be
expressed in terms of an
equation describing the
mode of drug action.
Most graded reactions are
consistent with the
sigmoid form, approaching
asymptotically to a “floor”
and a “ceiling.”

25. However, unlike the all-or-none response, where the number exposed to
treatment is known or can be estimated readily from control groups, the amount
of reaction representing 100 % of a graded response would need to be
determined experimentally to solve either formula.
By GRA, potency of a test agonist is determined by comparing its mean response
to standard mean response.

26. This process is known as ‘analytical dilution assay’. (Serial dilution of standard/test
drug) .
This assay simply depends on the several graded responses by exponential increase in
the test dose and which is compared with the standard graded dose response.
GRA is simplest way of determining potency of a test drug because it does not require
statistical analysis.

28. Bracketing Assay
This assay is preferred when
test sample volume is too
small. It is the simplest way of
GRA, in which single or few
response (s) is taken by using
any test drug concentration.
Consequently, this response is
bracketed between two
responses (one higher and
one lower) of the standard
drug.

29. Then the potency of the test drug is directly
calculated from concentration of standard drug or
by interpolation through dose response curve.
Limitation of the assay is poor precision
and reliability and also unable to calculate
error.

31. Matching Assay
Comparison of potency
between the unknown and
standard drug is done by
trial and error method.
Important part of this
method is that response is
matched at only one dose,
so it does not needs dose
response curve of test
compound.

32. It requires very small sample
volume, whereas meanwhile having
several disadvantages such as it is
purely subjective, experimental error
is not excluded out and there is no
sign of parallelism as it lacks dose
response relationship.
It requires most sensitive tissue, so
tissue selection is the most
important aspect in this assay.

34. Interpolation Assay
• This method depends on the assumption of dose response curve.
Concentration of unknown is interpolated from the dose response curve
graph.
• At the first step DRC of the standard drug is plotted then single or few
responses of the test drug are plotted.
• The dose of the test drug which comes at the linear log dose-response
relationship is interpolated from the dose response plot.

36. Multiple Point
Assay
• This methods are not ideal because of lack in
sensitivity, accuracy and might involve many
methodological errors such as tissue
sensitivity error, variable temperature error,
dilution error, etc.
• So, to correct the above mentioned
limitations, graded response assays are
preferred.
• Repeated response recording in graded
response assays minimize the tissue
sensitivity error and improve the
methodological errors.

37. These assays are performed by
the selection of 1 or more dose
responses of test compound
and these responses are
compared with 2 or more
responses of standards.
The selection of the test doses
must be in the linear portion of
the dose response plot of
standard compound, i.e.
between 25 to 75% (Dose
discrimination is better at these
portions).
Concentration of T Dose of ‘S’ × Conc. of ‘S’
Dose of ‘T’

39. Three-point assay
• Method depends on the latin square
randomization of total three responses selected
from DRC prepared for standard as well as test (2
response from standard and 1 response from
test: response selection is made between 25-75%
of response).
• For many bioassay, dose response data can be
transformed to generate log dose transformed
response lines to yield as extended a linear range
as feasible.
• Estimates of relative potency are then obtained
as the displacement of parallel log dose response
lines of standard and test compound.

41. Note: There are several factors plays role during the experiments such as
biological environmental or methodological factors. So, there may chances of
some error present during the bioassay.
Concentration of unknown compound = “x” times more concentrated than standard
compound
Where, S1 and S2 = Length of standard dose response selected between 25- 75%
T = Length of test dose response selected in between of two standard response
s1 and s2 = Standard drug dose which came in contact with tissue and given the response
‘S1’ and ‘S2’ respectively
t = Test drug dose which came in contact with tissue and given the response ‘T’

42. Four-point assays
• Method is same as 3-point assay,
only difference is that in this
experiment responses are selected; 2
responses of standard and 2 of test
from the DRC for the consecutive 16
response of Latin square
randomization as shown below in
figure.
• This procedure is more sensitive than
3-point assay and reduces the error
or variability.

44. • Where, S1 and S2 = Length of standard
dose-response selected
• T1 and T2 = Length of test dose response
selected
• s1 and s2 = Doses which produces mean
response of S1 and S2 respectively
• t1 and t2 = Doses which produces mean
response of T1 and T2 respectively

45. • Note: There are several factors plays role
during the experiments such as biological
environmental or methodological factors.
So, there may chances of some error
present during the bioassay.

46. 6-point and 8-point Assay
• These methods of bioassays are generally not adopted for the experiment
purpose because of the time-consuming lengthy procedure.
• The responses obtained for the 6-point is ‘36’ and ‘64’ for 8-point.
• But, the advantage being reduced error and variability of the procedure
over other methods due to the large number of responses and hence have
greater specificity.