BIOASSAYBy :Dr. Sumit Kumar Mahato Junior Resident( Academic)Guide: Dr. Uma Shanker Pd. Keshri Associate Professor Department of Pharmacology RIMS, Ranchi

An assay is an investigative (analytic) procedure in laboratory medicine, pharmacology, environmental biology, and molecular biology for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of a target entity (the analyte) which can be a drug or biochemical substance or organic sample

Assay

Chemical Assay Immunoassay Bioassay

Types Of Assay

Chemical Assay: It is the study of the separation, identification, and quantification of the chemical components of natural and artificial materials. Immunoassay:A technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample.

Comparative assessment of relative potency of a test compound to a standard compound on a living or biological tissue.

Quantitative measurement of the amount of active principle or substance in a pharmaceutical preparation or biological material using a suitable biological system

Introduced by Paul Ehrlich - biostandardization of Diphtheria antitoxin

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Bioassay

Comparison Of Chemical & Bioassay

BioassayBioassay

Chemical Assay Less Precise More time consuming More expensive Active constituent &

structure not known. More sensitive More men power

Required Difficult to handle

More Precise Less time consuming Less expensive Active constituent &

structure fully established.

Less sensitive Less men power

required Easy to handle

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Synonyms

Biological assay

Biometrics

Biological standadizatio

n

Bio-standadizati

on

To compare the test substance with the International Standard preparation of the same

To find out how much test substance is required to produce the same biological effect, as produced by the standard

Activity assayed should be the activity of interest

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Principles of bioassay

Standard & test sample - similar pharmacological effects & mode of action

Both should be compared for their established pharmacological effect using specified technique

Ex: *Ach – contractile response on frog rectus abdominis muscle

*Histamine – contractile response on guinea pig ileum

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Contd..

Problem of biological variation must be minimized

Experimental conditions - kept constant

Animals - same species, sex and weight

Number of animals - large enough to minimize error (individual variation)

Isolated preparations - sensitive

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Contd..

No chemical method has been developed

Chemical assay is too complex /not sensitive enough to measure (ex: insulin, Ach)

To measure the pharmacological activity of new or chemically undefined substances

For biological standardization of drugs obtained from

natural sources as these cannot be obtained in pure form. Eg: Oxytocin,Vasopressin,Insulin,Heparin..

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Indications of bioassay

To compare the strength of a drug obtained from various sources due to different compositions

Chemicals with similar structure, but different biological activity

Chemical structure of the active principle is unknown

Chemical structure known; cannot be actively purified. Eg: Peptide hormones

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Contd…

Sensitivity Specificity Repeatability Reproducibility Precision Accuracy Stability – tissue has to stay

“bioassay-fit

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Characteristics of a good assay method

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Bioassay can be performed on

• Intact animalsInvivo

• Isolated tissuesInvitr

o

WHOLE ANIMALS

Nor Adrenaline – Cat Cardiac Glycosides – Guinea Pig Insulin – Mice Estrogens – Ovariectamised Female Rat

ISOLATED TISSUE

Acetyl Choline – Frog Rectus Abdominus muscle Histamine – Guinea Pig ileum Adrenaline – Rat uterus Oxytocin – Rat uterus oestrogen primed

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Types of BioassayQualitative bioassayIs used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity.Eg: Arnold Adolph Berthold's famous experiment on castrated chickens. This analysis found that by removing the testes of a chicken, it would not develop into a rooster because the endocrine signals necessary for this process were not available.Quantitative bioassays involve estimation of concentration/potency of a substance by measurement of the biological response it produces. These bioassays are typically analyzed using the methods of biostatistics

1.Direct end point assay (DEPA)2.Quantal assay (all or none assay)3.Graded assay : a) Bracketting assay b) Matching assay c) Interpolation assay d) Multiple point assay ( 3-point, 4point, 6-

point, 8-point).

Bioassay Classification

Principle: is to measure direct response of dose of standard and test preparation. The threshold dose required for response is determined for each experimental unit. Ratio between these doses estimates the potency of the test preparation relative to the standard.

Concentration of test= TDS/TDT×CSD Where, TDS= Threshold dose of standard TDT= Threshold dose of test CSD= Concentration of standard drugThreshold dose of standard = Total period of infusion × rate

of drug administration

Direct End Point Assay:

Advantage: Drug effects appear rapidly and are easily

recognised Drug effect is directly proportional to drug dose Rapid end-point detection.

Disadvantages: Only toxicity study or high dose study is possible Dose ranging study cannot be done

Quantal response – the unknown is compared with the standard with respect to potency which produces the quantal affect, i.e change is easily recognised sign or often death.

In a quantal assay there is use of dose response relationship. quantal response to a drug is obtained and percentage of positive response

at each dose is calculated. The response in quantal assay is varying, i.e some responses are irreversible

and hence animal used is once and some responses have no permanent effect and animal can be used in next experiment

e.g – Determination of LD50 Hypoglycemic convulsion in mice in the assay of insulin

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Quantal assay( All or None assay):

Graded response -  In these assays, as the dose increases there is an equivalent rise in response. The potency of a test agonist is determined by comparing its mean response to standard mean response.

This process is also known as “analytical dilution assay”

GRA is the simplest way of determining potency of a test drug because it does not require statistical analysis

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Graded assay

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Graded DRC

standard Test/unknown

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DRC & Log DRC

30%

70%

Sigmoid curveWide range of doses can plot

Rectangular hyperbola

• Potency• Efficacy• Slope of curve

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Used when test sample is too small. Comparison of potency between unknown and standard drug is done

by trial and error method. Response is matched at only one dose Does not need dose respone curve of test compound It require most sensitive tissue Concentration of test= (Dose of standard/ Dose of test)× conc. Of standard

Disadvantage: Experimental error is not excluded out There is no sign of parallelism as it lack dose response relationship.

MATCHING ASSAY:

Used when test sample is too small. Single or few responses is taken by using any test drug

concentration. This response is bracketed between two responses one

higher and one lower of the standard drug. The strength of the unknown can be found by simple

interpolation of this bracketted response on dose axis

Bracketing Assay

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Bracketing or Direct Matching

less accurate,time consuming, troublesome cannot get exact match of response quantitative difference b/w test & standard not obtained

Disadvantages

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standard Test/unknown

A log dose-response curve is plotted with the standard, Single or few responses of test drug are plotted. The dose of test drug which comes at the linear log dose-

response is interpolated from the dose respone plot

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Interpolation assay

100

50

0 x x1

Interpolation

standard%

RESPONSE

LOG DOSE

Sensitivity of tissue is 1st determined by prior plotting of a conc-response curve with known agonist

Dose can be plotted even if it varies over thousand fold range

Error is normally distributed

Advantages

Sensitivity of tissue changes with time

Timing of doses not taken into account

Variation in mode of application of drugs

Disadvantages

Responses are repeated several times and the mean of each is taken

Chances of error are minimized 3 point method - 2 doses of std+1 dose of test 4 point method - 2 doses of std+2 doses of test 6 point method - 3 doses of std+3 doses of test

Latin square method of randomization to avoid any bias

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Multiple point assays

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3 - point assay t s1 s2s1 s2 ts2 t s1

3 cycles

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3-POINT ASSAY

%RESPONSE

LOG DOSEs1

T

t

S2

s2

S1

s1 s2 t

t s1 s2

s2 s1 t

t s2 s1

Latin square design

• Mean responses of these 3 sets plotted• Log potency ratio (M) = (T-S1÷ S2-S1)× log d where, d – dose ratio = s2/s1• Strength of unknown = s1/t × antilog of M S1, S2- length of standard dose response selected

between 25-75% T-length of test dose response selected in between of

two standard responsess2/s1 -standard drug dose which came in contact with

tissueand given the response S1, S2. respectively

t = Test drug dose which came in contact with tissu and given the response T 39

CALCULATION

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4 - POINT ASSAY

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4-POINT ASSAY

%RESPONSE

LOG DOSEs1

T1

t1

S2

s2

S1

T2

t2

s2 t1 t2 s1

t1 t2 s1 s2

t2 s1 s2 t1

Latin square designs1 s2 t1 t2

• Mean responses of 4 sets plotted• Log potency ratio (M)

(T2-S2)+(T1-S1) × Log d (S2-S1)+(T2-T1) where, d-dose ratio = s2/s1

• Strength of unknown = s1/t1 × antilog of M

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Calculation

3+3 dose assay 3 conc each of std & test drug are used 6 sets of experiments using 6 doses in each

set More time consuming,lesser in use Reliability is excellent

Six point assay

to measure the pharmacological activity of new/ chemically undefined substances

to investigate the function of endogenous mediators

to measure drug toxicity and unwanted effects

to measure the conc of drugs and other active substances in the blood or other body fluids

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Uses of Bioassay

Determination of potency, ED50/LD50 of drugs

New drug development

Measure clinical effectiveness

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• Biological variation• Troublesome• Time consuming• Expensive• Less accurate than physico-chemical

methods

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Drawbacks

Successful tool in estimation & discovery of biologically active substances

Sensitivity & Specificity – important tool in pharmacology

Conclusion