www.sankarpharmacology.wordpress.com UMA SANKAR GORLA M.Pharm., M.B.A.

umasankargorla@gmail.com Asst. Professor, Dept. of Pharmacology, VNIPS

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BIOASSAYS

BIOASSAY :

It is also known as Biological assay or Biological standardisation.

It is defined as the estimation of potency of an active principle in a unit

quantity of test preparation using living tissue/ microorganisms/ immune cells/

whole animal by comparing with that of the standard drug (International Units)

by determining its pharmacological action.

PRINCIPLE OF BIOASSAY :

The basic principle of bioassay is to compare the test substance with that

of the standard preparation of the same and to find out how much test substance

is required to produce the same biological effect as that of the standard by using

a suitable biological system at uniform experimental conditions.

Graded Response:

Response is directly proportional to dose.

Response lie between no response & maximum.

Quantal Response:

Response – All or none.

Either no response or maximum response.

www.sankarpharmacology.wordpress.com UMA SANKAR GORLA M.Pharm., M.B.A.

umasankargorla@gmail.com Asst. Professor, Dept. of Pharmacology, VNIPS

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MERITS:

Bioassay is the only method of assay, if when

a) The active principle of the drug is unknown or can’t be isolated.

Ex: Insulin posterior pituitary extract.

b) Chemical method is not available; if available, it is too complex or

insensitive or requires high doses of test sample.

Ex: Insulin Acetylcholine.

c) Chemical composition is not known.

Ex: Long acting thyroid stimulants.

d) Chemicals composition of the drug differs but has same pharmacological

action.

Ex: Cardiac glycosides (Na+/K+ pump Inhibitor) and neurotransmitters

(Catecholamine's).

e) When the quantity of sample is too small.

DEMERITS:

• Less accurate

• Less elaborate

• More laborious

• More troublesome.

• More expensive.

• Time consuming.

www.sankarpharmacology.wordpress.com UMA SANKAR GORLA M.Pharm., M.B.A.

umasankargorla@gmail.com Asst. Professor, Dept. of Pharmacology, VNIPS

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METHODS OF BIOASSAY:

I) Direct methods

a) End Point Method.

b) Matching Method.

c) Bracketing Method.

II) Indirect Methods

a) Graphical/ Interpolation Method.

b) Multiple Point Method.

i) Three point Bioassay.

ii) Four point Bioassay.

iii) Six point Bioassay.

www.sankarpharmacology.wordpress.com UMA SANKAR GORLA M.Pharm., M.B.A.

umasankargorla@gmail.com Asst. Professor, Dept. of Pharmacology, VNIPS

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End Point Method of Bioassay:

This method is used to determine the threshold does of test drug

producing a positive effect by comparing with the standard drug.

Ex: Bioassay of Digitalis in Cats.

Cat

Anaesthetised with Chloralose

B.P. is recorded

Standard/Test drug Infused slowly

At zero B.P., Heart stops beating.

Conc. of drug infused is noted.

Threshold does of standard

Conc. of Test = ---------------------------------- X Conc. of standard

Threshold does of Test

Matching method of Bioassay:

In this method, the responses obtained from standard drug and test drug

are matched by a trial and error process until they produce equal effects.

Advantage:

It does not depends on dose – response relationship.

Disadvantage:

• It is purely subjective.

• Experimental errors cannot be determined.

• Qualitative differences between standard and test drug.

Ex: DRC of Acetyl choline by matching method using frog rectus

abdominus muscle.

Note: Test is matched with either S1 or S2 by increasing or decreasing the

volume of test does. Here the test is matched with S2 response.

S2 response = T response at 1.3 ml

16 mg = 1.3 ml of Test.

:. 1 ml of Test = 16/1.3 = 12.30 mg/ml

Bracketing Method of Bioassay:

In this method, a constant does of the test is bracketed by varying doses

of standard i.e., by increasing or decreasing the doses of standard (S1 & S2) till

all three equal responses are obtained.

Advantage:

It does not depended on dose-response relationship.

Disadvantages:

• It is purely subjective

• It occupies large area of drum

• Experimental errors cannot be determining.

Ex: bioassay of histamine on guinea pig ileum using bracketing

Method

Responses

S1 = T = S2

10µg = 1ml = 10µg

Test 1ml = 10µg

S1 + S2

Potency of the Test = ---------- µg/ml

2

9+10

= ---------- µg/ml

2

19

= --------- µg/ml

2

= 9.5 µg/ml

Graphical / Interpolation method:

In this method, dose response curve (5-6 responses) of the standard are

taken and the height of contraction is measured and plotted against the log-

dose.

Then the response of the test sample is taken, height of contraction is

measured and the concentration is measured by interpolating the standard

curve.

Advantages:

• Less time consuming

• More reliable method

• Based on dose-response relationship

• Less chances of errors

Limitations:

The response of test must lie within 20 – 80% responses of the standard.

Concentration of Test = Log dose 0.75

= Antilog 0.75

= 5.62 µg/ml

Three point bioassay:

In this method, dose response curve is recorded by using graded doses of

the standard until the ceiling effect.

Then select two responses of the standard i.e. 20% response & 70%

response and named as S1 & S2 respectively.

Select one test dose which will give response in between 20% & 70% of

the standard [to maintain close bracket].

Finally record the responses of the S1, S2, and T in the following sets

Set-1 S1 S2 T

Set-2 S2 T S1

Set-3 T S1 S2

www.sankarpharmacology.wordpress.com UMA SANKAR GORLA M.Pharm., M.B.A.

umasankargorla@gmail.com Asst. Professor, Dept. of Pharmacology, VNIPS

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n 1 T - S1 n 2

Conc. of Unknown = -------- X antilog ---------- X log ------- X Cs

t S2 - S1 n 1

Where

n1 = Vol. of lower standard dose (S1)

n2 = Vol. of higher standard dose (S2)

t = Vol. of Test dose (T)

S1 = Avg. response of lower standard dose (n1)

S2 = Avg. response of higher standard dose (n2)

T = Avg. response of test dose (t)

Cs = Conc. of standard.

Response (cm)

S1 S2 T

Set-1

Set-2

Set-3

Avg

Four point bioassay:

In this method, dose response curve is recorded by using graded doses of

the standard until the ceiling effect is obtained.

Then select two responses i.e. 20% response and 70% response and

named as S1 S2 respectively.

Select two test doses T1 & T2 which will give responses in between 20%

&70% responses of the standard (to maintain close bracket).

Finally record the responses of S1, S2, T1,& T2 in the following sets.

Set-1 S1 S2 T1 T2

Set-2 S2 T1 T2 S1

Set-3 T1 T2 S1 S2

Set-4 T2 S1 S2 T1

Response (cm)

S1 S2 T1 T2

Set-1

Set-2

Set-3

Set-4

Avg.

n 1 (T1 - S1)+(T2-S2) n 2

Conc. of Unknown = ---- X antilog ---------------------- X log --- X Cs

t1 (T2-T1)+(S2 - S1) n 1

Where

n1 = Vol. of lower standard dose (S1)

n2 = Vol. of higher standard dose (S2)

www.sankarpharmacology.wordpress.com UMA SANKAR GORLA M.Pharm., M.B.A.

umasankargorla@gmail.com Asst. Professor, Dept. of Pharmacology, VNIPS

18

t1 = Vol. of lower Test dose (T1)

S1 = Avg.response of lower std. dose (n1)

S2 = Avg. response of higher std. dose (n2)

T1 = Avg. response of lower test dose (t1)

T2 = Avg. response of higher test dose (t2)

Cs = Conc. of standard.