BioassaysDR . PRAVEEN THAGGIKUPPE KRISHNAMURTHYASS ISTANT PRO FESSO RDEPT. OF PHARMACOLO GY, J SS COLLEGE OF PHARMACY , O OTYEmai l : p raveen tk7812@gmai l . com

Bioassay Bioassays are procedures that can determine the concentration or purity or biological activity of a substance (such as vitamin, hormone, and plant growth factor) by measuring its effect on a organism, tissue cells, enzymes or the receptor.

Bioassays, as compared to other methods of assays (e.g. chemical assay) are less accurate, less elaborate, more laborious, more troublesome and more expensive

Indications for bioassaysBioassay is the only method of assay if;

1. Active principle of drug is unknown or cannot be isolated, e.g. insulin, posterior pituitary extract etc.

2. Chemical method is either not available or if available, it is too complex and insensitive to lower dose e.g. Insulin, acetylcholine, histamine.

3. Chemical composition of drug varies but has same pharmacological action (e.g. cardiac glycosides isolated from different sources, catecholamine's, etc.)

4. When the quantity of the sample is too small

Indications for bioassays……

5. To estimate the concentrations of the active principles present in the tissue extracts (Active principle cannot be isolated, e.g. insulin, posterior pituitary extract etc.)

6. To measure the pharmacological activity of new or chemically unidentified substance

7. To measure the drug toxicity

8. When bioassay is more sensitive than the chemical assay e.g. Histamine

Principles of Bioassay1. The bioassay involves the

comparison of the main pharmacological response of the unknown with that of standard.

2. The reference standard and the test sample should share the same mode of action so that their LDR curves run parallel and their potency ratios can be conveniently compared.

3. The test solutions and the reference standard should be compared for their established pharmacological effects using a specified pharmacological technique (appropriate model) (Ex: Ach-frog rectus abdominus muscle, Histamine- guinea pig ileum)

4. The method selected should be reliable, sensitive, reproducible and should minimize errors due to biological variation and methodology (ex: 3-point vs matching point bioassay)

5. Hence, the animals used should always be of the same species, sex and weight and, if isolated preparation is used it should be sensitive.

6. The number of animals should be large enough to permit statistical analysis and experimental conditions should always be kept constant

TypesBioassay

Quantal bioassay Graded bioassay

Matching Bioassay

Interpolation Bioassay

Bracketing Bioassay

Multiple Point Bioassay

Quantal assayA quantal assay involves an "all or none response“

The response is either Positive or Negative, there is no intermediate response

Example: Insulin induced hypoglycemic convulsive reaction; The cardiac arrest caused by digitalis; Calculation of LD50 in mice or rats

The quantal method though not precise. It is still employed for bioassay of substance in the following ways:

a. Comparison of threshold response or

b. Comparison of effective dose (ED50) or median lethal dose (LD50)

Graded assays Graded assays are based on the observation that there is a proportionate increase in the observed response following an increase in the concentration or dose.

Here the responses to varying doses of the drug are graded and measured

Bioassay of histamine on guinea pig ileum

A graded bioassay can be performed by employing any of the below-mentioned techniques.

◦ Matching Bioassay◦ Interpolation Bioassay◦ Bracketing Bioassay◦ Multiple Point Bioassay (i.e.-Three-point, Four-point and Six Point Bioassay)

The choice of procedure depends on:

◦ the precision of the assay required

◦ the quantity of the sample substance available

◦ the availability of the experimental animals.

Matching Bioassay It is the simplest type of the bioassay.

T- 0.2 ml S- 0.8 S- 0.4 S- 0.3 S- 0.3

Matching assay

Advantages and limitations

This assay is applied when the sample size is too small. The method is particularly useful if the sensitivity of the preparation is not stable.

It occupies a larger area of the drum as far as tracings are concerned.

The match is purely subjective, so chances of error are there and one cannot determine them.

It does not give any idea of dose-response relationship.

Bracketing bioassay

S1

S2

T

S1

S2

T

ConcentrationRe

spon

se

This assay is applied when the sample size is too small.

The method is particularly useful if the sensitivity of the preparation is not stable.

It does not give any idea of dose-response relationship.

Interpolation assay It is based on the principles of log dose response (LDR) curves

0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8

Log dose

% m

axim

um re

spon

se

0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8

Log dose

% m

axim

um re

spon

se

Test responses

Standard DRC

All the three methods described above are not reliable because these do not take into account:

◦ Changes in the sensitivity of the tissue with respect to time

◦ The timing of the doses

◦ Variations in the mode of application of the drugs, and are based on the application of a single dose (few doses) and hence cannot be statistical evaluation

Multipoint assays Three point assay (2+1 dose assay)

Four point assay (2+2 dose assay)

Six point assay (3+3 dose assay)

These methods take into account:

◦ Changes in the sensitivity of the tissue with respect to time

◦ The timing of the doses

◦ Variations in the mode of application of the drugs, (Doses are repeated and hence can be statistical evaluation)

◦ These methods employ the Latin square design and the responses are recorded in a random fashion

The precision, reliability and reproducibility of these assays methods are very high

S1 S2 TS2 T S1T S1 S2

Three point assay (2+1 dose assay)

Take the DRC of the standard

Select two doses of standard (s1 and s2) whose responses (S1 and S2) lie within the linear portion of LDR curve.

In addition, the s1 and s2 dose are selected such that their concentrations are in simple ratio (ex. 1:2 or 2:4) (0.1 ml and 0.2 ml)

Subsequently, a dose (t) of the test sample which produces a response (T), in between S1 and S2 is found by trial and error method.

0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8DRC of standard

s1

0.1 ml

s2

0.2 ml

t0.2ml

The responses of the selected doses were recorded in a randomized fashion using latin square design

s1, s2, t, s2,t,s1, t,s1,s2

Here none of the sequence is repeated, i.e. the doses have been randomized to reduce error

s1 s2 ts2 t s1t s1 s2

s1 s2t s2

t s1t s1 s2

Calculation Method-1 (Graphical method)

The respective percentage mean responses are then plotted on the graph paper against log dose of standard

The concentration of the unknown is determined by interpolation

S1

S2

T

Concentration

Resp

onse

Method-2 (Formula method)

◦ Conc. of std – 10 mcg/ml◦ Dose s1= 0.1ml, s2= 0.2, t= 0.25 ml ◦ Response (Average) S1= 40 mm, S2= 80mm, T=60 mm

◦ Unknown concentration=s1/t × antilog {[(T-S1)/(S2-S1)] × log (s2 /s1)} × conc. of std

◦ =5.6 mcg/ml

Four point assay Record the DRC of both test and standard

Select two doses of standard (s1 and s2) (responses S1 and S2 should lie b/w 25 and 75 %)

Select two responses of test such that T1 lies between S1 and S2 and T2 should be greater than S2

Record the responses with the selected four doses using the latin square design

0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8DRC of standard

s1 s2 t1 t2

s2 t1 t2 s1

t1 t2 s1 s2

t2 s1 s2 T1

Calculation Method 1 (graphical method)

Method 2 (Formula method)

• Unknown concentration=s1/t1 × antilog {[(T2-S2)+(T1-S1)/(S2-S1) +(T2-T1)] × log (s2 /s1)} × conc. of std

Thank you