Biological assays/Bioassays

-A type of scientific experiment:

Conducted to measure the effects of a substance on a living
system and are essential in the development of new drugs
and in monitoring environmental pollutants.
or
Determination of the chemical & biological agents such as
the drugs, hormones, vitamins, toxins & anti-toxins by means of
the biological indicators such as blood pressure, blood glucose,
muscle contraction & the inhibition of the growth of the micro-
organisms.


-Animal, organ, tissue, cell lines/microbes or sub cellular
parts are used for the purpose.

-Can be divided into two categories:

i)Qualitative (Measures the physical effects)
e.g. abnormal growth or deformity etc.

ii)Quantitative

-Concentration or potency of a substance by measurement
of the biological response that it produces.




Conditions of usage:

i)Determination of the drug potency.

ii)Screening of the new pharmaceutical agents.

iii)Determination of the therapeutic advantages of the one
drug with another drug.

iv)To establish the structural activity relationships.

v)Determination of the pharmacological activities of the
new drugs.



iv)To determine the specificity of a compound to be used
e.g. Penicillins are effective against Gram+ve but not
on Gram-ve

v)Chemical composition of samples are different but have
same biological activity Or activity may vary even
though the chemical is the same ( e.g. due to optical
isomerism)



No alternatives in the following conditions:

i)For the drug whose active principles are unknown.
(e.g. Extracts, crude drugs)

ii)For the drugs chemical methods are not available.
(e.g.Cyanocobalamin in some dosage forms)

iii)For the samples which are available in minute
quantities.




Principle:

It is to compare the sample substances with the
international standard preparations of the same
&
to measure the potency of the sample that is required to
produce the same level of biological effect as that of the
standard drug.











Types of bioassays :

i)Quantal assays(end point method)
ii)Graded response assays

i)Quantal assays:

-To draw or elicit an all or none response in the different
animals such as the following:

a)Digitalis induced cardiac arrest in the guinea pigs.

b)Digitalis induced head drop in the rabbits.
















ii)Graded response assays:

-Varying doses are given to one animal.

-As the dose increases there is an equivalent rise in
response.

-The potency is estimated by comparing the test sample
responses with the standard response curve.


















Variation of bioassays based on biological systems:

i)Animal(mouse, rat, guinea pig, rabbits etc)

ii)Micro-organisms or cell based assay (using microbes
like bacteria, fungi or cultured cells for antibiotics
& growth stimulants/suppressants.

iii)Plant(using plant constituents to evaluate a sample)

















i)Animal(mouse, rat, guinea pig, rabbits etc)

Bioassays for agonists:

a)End point method(based on quantal effect):

-The threshold dose producing the therapeutic effect is
measured on the each animal.

-Then comparison is made between the average results of
the two groups of animal.

















-Sp./Std. are carried out simultaneously.

-Finally potency is calculated from the following formula:

Conc. of the unknown= threshold dose of Std. X Conc. of Std.
threshold dose of Sp.





























b)Matching method(based on the graded response):

-The constant dose of the test is bracketed by varying
doses of the standard until the exact match is observed
between the sample & the standard.

-Response of the standard is measured at 20% & 70% of
the sample.

-The test will be repeated by increasing or decreasing the
standard doses until the three equal responses are
obtained.









































The concentration of the sample is determined by the
following formula:

Concentration of the sample= dose of Std.xConc.of Std.
dose of the Sp.

c)Graphical method(based on the graded response):

-5-6 responses of standard graded doses of the standard
drug are taken & the two doses of the sample drug are
taken.

-The height of the peak is plotted against the log dose.









































-The dose of the sample is directly taken from the graph.

Conc. of the Sp.= [n
1
/t] x antilog{[(T-S
1
)/(S
2
-S
1
)] x [log n
2
/ n
1
]} x C
s

Where, n
1
is the lower standard dose; n
2
is the higher
standard dose; t is the test dose, S
1
is response of lower
standard dose; S
2
is the response of the higher standard
dose, T is the response of test; C
s
is the concentration of the
standard.











































ii)Micro-organisms or cell based assay:
-It is used to evaluate the potency of the formulations used.
(to follow purification process, its distribution within the blood & tissues when given to
animals & finally human. Monitor of the antibiotics in blood & urine in a trace amount)

-Used in case of unavailability of physical or chemical
method.
-Is performed using a suitable test organism.

-Most micro-biological assay procedures are highly
sensitive(up to 0.1mcg) & selective.

-Sensitivity & selectivity are to measure the characteristic
activity of biological products.









































Assay techniques:

i)Plate diffusion

ii)Tube-dilution









































i)Plate diffusion:

-The agar plates are inoculated with a test organism.

-Measured amounts of test substance are introduced into
agar plates.

-It diffuses into the agar & produces zones of inhibition.

-The diameter of the zones may be plotted against log
potency or conc. for a standard.













































-By interpolation, potency of the test material can be
determined.

-Suitable for antibiotic potency test.

ii)Tube dilution:

-Suitable dilutions of sample are introduced into an
appropriate nutrient medium.

-Suppression or stimulation of growth takes place.

-Suitable for the determination of vitamin B & amino acids.















































-Measurement is done by nephelometry, titration or
gravimetry.
Applications:
---------------------------------------------------------------------------------------------
Drug name Activity assayed Experimental animal/microbes--
--------------------------------------------------------------------------------------------
Digitalis Fall in BP Guinea pig blood pressure
Adrenaline Rise in BP Blood pressure in the spinal
cat
Acetylcholine Contractile effect Isolated rectus of the frog
Heparin Prolongation of the
Blood clotting time Whole blood of OX
Insulin Lower of the BL. Rabbits
Glucose level
Oxytocin Ejection of milk Female rabbits
Erythromycin Growth inhibition Bacillus pumilus/
Micrococcus luteus