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A SEMINAR ON BIOASSAY:PRINCIPLES OF

BIOASSAY
AAFTAB ANWAR
LUQMAN COLLEGE OF PHARMACY
DEPARTMENT OF PHARMACOLOGY
GULBARGA
Selection of Method:-

Quantitative estimation can be done
by..
.
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 by measurement of biological
response it produces.
i.e. Observation of pharmacological effects on
[1] living tissues, or cells
[2] microorganisms
[3] animals

Also known as BioStandardization.

Bioassay generally
employed on..
Chemical assay is not available.
Quantity of sample too small.
Estimate concentration of active principle
in tissue extract.e.g..insulin.
Estimate pharmacological activity of
unidentified substance,
Measure drug toxicity,
Diagnosis & research.
Dose of drug required to produce
therapeutic effect.
If active principle of drug is unknown,
e.g. insulin.
Chemical method not available.
Chemical composition not known.
Purification for chemical assay not possible.
To ascertain the potency hence served as
quantitative part of screening procedure.
Investigate function of endogenous mediators.
Measure drug toxicity & unwanted effects.
Principles of Bioassay:
1)All bioassays(lab.studies , toxicity
studies , clinical trials)must be
comparative against a standard drug or
preparation.


Way of minimizing error.
Representative of substance serves as basis
for comparative measurement of activity.

In India maintained & distributed by:

Central Drug Research Laboratory,Kolkata.
Central Research Institute,Kasauli.

Standard Preparation:-
2)Standard & new drug should be as
far as possible identical to each other.


So,dose response curve will have
same slope & parallel.


3)Method of comparison preferably(not
essentially) test therapeutic property
of drug.

4)Method should estimate as far as
possible the error due to biological
variation.


[1] Quantal Assays [ Direct endpoint ]
Elicits an All or None response in different
animals
E.g..
Digitalis induced cardiac arrest in guinea pigs
hypoglycaemic convulsions in mice.
Digitalis induced head drop in rabbits
Calculation of LD50 in mice or rats
[2] Graded Response Assays [mostly on
tissues]
Graded responses to varying doses
Unknown dose response measured on same
tissue


Graded:
Here proportionate increase in responses due
to increase in dose or concentration.

E.g. contraction of smooth muscle for
histamine assay.
I t can be done by following
methods..
MATCHING BIOASSAY:-Firstly
responses of test is taken & is
matched with response of standard
drugs.
Done till a closed matching is
observed.
Corresponding concentration
calculated.
Employed for small sample size.
Concentration response curve of
standard established.
2-3 responses of testis recorded.
Selection such that they lie on
LINEAR PORTION of CRC of
standard drug.
Precision & reliability is better.
I NTERPOLATI ON:-
MULTI PLE POI NT BI OASSAY
Three point bioassay:- [2+1 dose assay] two standard & one test responses are
taken down.
Fast & convenient
Procedure [E.g. Ach bioassay]
Log dose response [LDR] curve plotted with varying conc of std Ach solutions
and given test solution
Select two std doses s1& s2 [ in 1:2 dose ratio] from linear part of LDR [ Let
the corresponding response be S1, S2]
Choose a test dose t with a response T between S1 & S2
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 = dose ratio]


Four point bioassay:-two standards & two
test responses are recorded.

Two responses of standard should lie on
linear portion of CRC in ratio of 1:2.

Test response is by trial & error method.

Employing statistics responses are
recorded.

Most common method used.
SIX POINT BIOASSAY:-
Three concentration of standard
& test are used.



More time consuming method.
BRACKETTI NG METHOD:-
Used when test sample is too small.

Response of test is bracketed between
two responses ( greater & smaller ) of
standard substances.

Precision & reliability is poor.
MERITS:
Biological products like toxin,anti toxin,sera can
be conveniently assayed.

Measure minute(nano mole & Pico
mole)quantities of active substances.

Can detect active substance without prior
extraction or other treatment.
DEMERITS:
Key problem is variability in response.
Large number of animal to be used.
Expertise in experimental
design,execution of assay & analysis of
data required.
Leads to expensive & time consuming.
Time related changes ins sensitivity of
test organ.
Tachyphylactic responses of substance
being assayed.
CALCULATI ON OF DOSES:-
Expressed in mg/kg.
Common route of administration
INTRAPERITONIAL route. as..
Quick onset of action, better
absorption.
Volume of injection in rat-0.5 ml/100g
of body weight, in mice 1ml/100g of
body weight.
Make one ml more then required.

Barbiturates mostly used.

E.g. Phenobarbital sodium(NEMBUTAL).

Produces anesthesia in dose of 35-45mg/kg.

Give intraperitonialy/intravenous routes

Duration 45-60 min.

Others are chloralose (80-100mg/kg,ip or iv)&
urethane (1-1.5g/kg,ip or iv).
LABORATORY ANAESTHETI CS
REFERENCES:
Elements of
pharmacology,Goyal,R.k.
Essentials of
pharmacotherapeutics,Barar,F.S.K.
Hand book of experimental
pharmacology,kulkarni.s.k.

ANY QUESTIONS
PLZ.