BIOASSAY ANSWERS (SET 1)

Bioassay Definition
Estimation of the concentration /or potency of a substance by measurement of
the biological response that it produces.
Uses:
1. To measure the pharmacological activity of new or chemically undefined
substances.
2. To investigate the functions of endogenous mediators.
3. To measure drug toxicity and unwanted effects ED50 and LD50.
4. Drug discovery: the ability of extracts of posterior lobe of pituitary to
produce rise in BP and contractions of uterus was observed in 1930s and
were found to be ADH and Oxytocin.
AIM: Comparing the activity of 2 preparations, a standard(s) and an unknown(u)
on a particular preparation.
Bioassay must provide an estimate of dose/concentration of unknown that will
produce the same biological effect as that of known dose/concentration of
standard.
Main problem with bioassay: biological variation.
M= potency ratio between S/U

M
Response
U

Log vol

In practice, most bioassays will give

results with 5% confidence limits lying within 20%.
Graded response / Assay
E.g.: change in BP, glucose concentration, concentration of strip of smooth
muscle.
The steepness of the DRC is a property of the test system and has nothing to do
with biological variation
Relates to size of responses in single biological unit.
All or none response / Discontinuous / Quantal
E.g.: death, loss of righting reflex
LD50, ED50, Polygenic Random variation
As the dose increases the response increases reflecting the extent of receptor
occupancy.

Threshold dose and ceiling dose

This allows us to compare therapeutic efficacy of various compounds.

[1]

Advantages of isolated tissue.

Several preparations can be tested from a single animal.
Relatively small amount of test material required.
Drug effect is tested directly without factors like A, D, M , E

[2]

Why ileum?
Relatively more resistant to trauma/handling
Easy to set up
Produce larger contractions than in duodenum and jejunum
Less mesentery
More receptors.

[3]

What is Bio phase?

Environment in which drug interacts with receptor without intervening
diffusion barriers. Here it is the environment within the inner organ bath
with tissue, physiological salt solution and drug.

[4]

What do you mean by analytical dilution assay?

Here the nature of test compound and the standard are same and the
concentration / potency of test is determined by dilution or concentration
with standard. Eg :

[5]

What is comparative assay?

Here the test compound is chemically different from the standard, but both
have similar activity. Eg: Histamine and Acetylcholine on G.pig tracheal
chain

[6]

[7]
i.
ii.
iii.

What is bio standardisation?

Comparison and adjustment of the strength of the sample with that of the
standard under controlled conditions.
Necessary to regulate doses of crude extract.
Eg: If we are preparing tetanus antitoxin then the activity of 1TU of our
antitoxin should be standardised with that of standard preparation in
National Serum Institute,Copenhagen (0.1547 mg)
(Bioassay = Measurement; Biostandardisation = Alteration)
Why Log Phase Response?
Results can be plotted (obtained) even with wide variation of doses over a
1000 fold range.
The middle portion of sigmoid is almost linear.
Error is distributed all through the graph independent of dose.

iv.

When two are more graphs are plotted middle portions will be almost
parallel hence can be compared easily.
[8]

[9]

What is pD2 value?

Another way of expressing potency.
It is the mean negative logarithm of molar concentration producing 50%of
maximal response.
Higher the pD2 value, Higher is potency.
What is pAx value?
Schild proposed it to express drug antagonism.
Negative log of molar concentration of an antagonist, which will reduce the
effect of a multiple dose X of an active drug to that of single dose.

[10]
Drugs that contract and relax guinea pig ileum?
Contraction
Relaxation/No effect
*5 HT
*Noradrenaline
*Histamine
*Adrenaline
*Acetylcholine
*Angiotensin
*Bradykinin
[11]
Disadvantages of Matching Assay:
It does not consider changes in tissue sensitivity with respect to time.
Purely subjective.
Timing of doses not taken into account.
It is based on application of few doses hence cannot be subjected to
statistical evaluation (error cannot be calculated).
[12]
Uses of salts in physiological salt solutions:
Na+
-carry the depolarising current essential for excitation.
ions
-provide adequate osmotic pressure
K+ ions -essential for bringing the process of relaxation to completion
Ca+ ions -for coupling the excitation with contraction
-stabilises membrane potential
Mg+ ion -reduce spontaneous contractility in mammalian tissue
NaH2PO4 -acts as buffer
[13]
Who discovered organ bath?
Organ bath is an assembly for recording contractions of isolated tissue.It
was first designed by Rudolph magnus in 1904
The present day organ bath was designed by Sir Henry Dale.
Student organ bath having two units of inner tissue bath (double unit
organ bath) was designed by Gaddum.

[14]
Information about drum
It is called Sherrington Drum
By convention, it rotates in a clockwise direction, hence all records to be
read from left to right.
Speed of drum
o *Guinea pig ileum-32 minutes per revolution
o *Frog rectus-128 minutes per revolution
[15]
How will you check pH? Why is it important?
The pH of salt solutions should be maintained between 7.3-7.4.
Ph is checked with Litmus paper or commercially available pH strips.
Prolonged aeration tends to alter pH
At lower pH the tonus of the tissue decreases, there by altering the effect
of drugs.
[16]
What are the uses of Aeration?
Provides oxygen to the tissue.
Facilitates diffusion of drug Stirring.
Maintains vitality of tissue and helps to overcome inertia.
[17]
What is Differential bioassay?
If more than one active substance is present then it is called Differential
Bioassay.
[18]
How will you manage Spontaneous Activity of tissue?
For frog rectus: 5-10 g/L of neostigmine can be added to the reservoir (salt
solution)
For guinea pig ileum:
o Atropine 1mg/L or
o Adrenaline (But there will be baseline shift) or
o Lowers the bath temperature
[19]
How will you prevent the friction between lever and drum ?
The writing point should be sharp
The paper should be smooth
Smoking should be thin and uniform
Vibrator can be fixed
[20]
Give example of microorganism used for bioassay.
Euglena gracilis is used for vit B12 bioassay.
[21]
Examples of isolated cells in bioassay
Isolated Leydig cells of testes are used in-vitro for bioassay of LH hormone.
Plasma LH has ability to stimulate testosterone synthesis.
[22]
Give an example of indirect bioassay?
If the potency of sample is estimated by comparing its log dose response
with that of standard then it is called indirect assay.
Old ex: ergot preparation, when injected in the white leghorn cock, there is
bluish discolouration of the comb, due to vasoconstriction. The colour

intensity varies with the dose and this effect may be employed for bioassay
of crude ergot preparation.
Q. Requirements:
High sensitivity
Specificity Eg: G.pig contracts with Acetylcholine and histamine, therefore
the tissue has be atropinised
Reproducibility
Accuracy
Stability: Tissue /animal should be bioassay fit for long time.
Q. Whole Animal Bioassays.
Substance
1.Vasopres
sin
2.Insulin
3.d-TC
4.Estrogen
5.Vit.D

Preparation
Increases BP in anaesthetised rat
Decreases urine output in hydrated rat.
Hypoglycemic convulsion or death in mice
Head drop in rabbit due to paralytic relaxation of skin of the
neck
Vaginal cornification in ovariectomised female rat
Alleviation of rachitic state in rat

Q. Sat. Assay
Assay: Estimation of amount or activity of an active principle in unit quantity of
preparation
Eg: Chemical assay, biological assay, immunological assay
Indications:
a. Chemical composition not known but substance has special biological
action. Eg: long acting thyroid stimulator.
b. Chemical assay is too complex or insensitive
c. Drugs differ in composition but have same pharmacological action eg:
digitalis glycosides from various sources.
d. Active principle is unknown /cannot be isolated easily.
Principles:
a) Effect produced should be same in all animal species.
b) Certain quantity of drug produces same degree of pharmacological
responses in the same animal provided other conditions remain constant
c) Reference standard must owe its activity to the principle for which the
sample is being bioassayed.
d) The activity assayed should be the activity of interest.
e) Problems of individual variation must be minimised or taken into account.
Indirect assay: potency of the sample is estimated by comparing DRC of
Standard
Direct assay: ED 50 and LD 50 calculated Eg: digitalis bioassay in guinea pig