Biological standardization (Bioassay)- Definition Bioassays are employed- BUT WHEN??? Applications of bioassay methods Principles of bioassay of drugs Standard preparation (Reference Standard) Types of bioassays- 1) Quantal response bioassay 2) Graded response bioassays a) Matching bioassay b) Interpolation bioassay c) Bracketing bioassay d) Multiple point bioassays Three-point bioassay Four-point bioassay Six-point bioassay Latin square designs- Multiple point bioassays Intact animal studies

1. Experiment No. 3
Introduction to principles of bioassay, its types
including advantages and disadvantages
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5
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2. Overview of Discussion
 Biological standardization (Bioassay)- Definition
 Bioassays are employed- BUT WHEN???
 Applications of bioassay methods
 Principles of bioassay of drugs
 Standard preparation (Reference Standard)
 Types of bioassays-
1) Quantal response bioassay
2) Graded response bioassays
a) Matching bioassay
b) Interpolation bioassay
c) Bracketing bioassay
d) Multiple point bioassays
 Three point bioassay
 Four point bioassay
 Six point bioassay
 Latin square designs- Multiple point bioassays
 Intact animal studies
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3. Biological standardization (Bioassay)-
Definition
The procedures by which the potency or the nature of the
substance (constitution) is estimated by studying its
effects on living matter.
Bioassays can be done by using-
1) An animal tissues or organs as in the case of use of guinea
pig ileum for the estimation of histamine, or
2) The intact animal as in the case of bioassay of insulin using
mouse or digitalis using guinea pig.
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4. Bioassays are employed- BUT WHEN???
 When a chemical assay for the substance is not available or the
substance gets inactivated by interacting with chemicals as the
case with hormones,
 When quantity of the sample is too small. In such situation, a
matching type of bioassay is conveniently done to compare the
biological response with the standard drug,
 To estimate the concentration of active principles present in the
tissue extracts, the endogenous mediators like acetylcholine
(ACh), serotonin or 5-hydroxytryptamine (5-HT), prostaglandins
(PGs),
 To measure the pharmacological activity of a new or chemically
unidentified substances,
 To measure drug toxicity, and
 When the bioassay is more sensitive than the chemical assay.
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5. Applications of bioassay methods
 Standardization of drugs of natural origin,
 Estimation of biologically active substances like ACh, adrenaline,
nor-adrenaline and serotonin in body fluids or tissue extracts,
 Screening of new compounds for biological activity. Even
synthetic products are subjected to these methods, as chemical
structures does not truly predict the pharmacological activity,
 Bioassay is employed if the drug is composed of a complex
mixture of substances of varying structure and activity, e.g.
digitalis, posterior pituitary hormones,
 Diagnosis and research- The concentration of gonadotropins in
the blood or urine may be estimated by injecting these fluids in
animals. (Chemical methods may be employed, if available), and
 Estimation of the dose of a drug required to produce a
therapeutic or toxic responses, e.g. ED50 (median effective dose)
or LD50 (median lethal dose).
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6. Principles of bioassay of drugs
Burn and Dale have enunciated certain principles for the conduction
of bioassay procedures. These principles must be followed for
obtaining valid results.
Principle 1) All bioassays (laboratory studies, toxicity studies, clinical
trials) must be comparative against a standard drug or
preparation. This is one way of minimizing error in bioassays due
to biological variation as the new sample is simultaneously
compared with the standard preparation.
Principle 2) The standard and the new drugs should be as far as
possibly identical to each other. If it is so, their dose response
curves will have the same slope and would be parallel and the
potency ratio would be constant all along the response levels.
However if the curves are not parallel, this statement is invalid.
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7. Principles of bioassay of drugs
Principle 3) The method for comparing unknown and the standard
drug should preferably (but not essentially) test therapeutic
property of the drug. Ideally, analgesic drugs should be tested for
analgesic activity, and anticonvulsants should be tested for
anticonvulsant activity.
However, it is not always possible to do so, e.g. for insulin, lowering
of blood glucose would be the ideal potency index, but the
commonly used method for the screening is insulin induced
hypoglycaemic convulsions in mice, in which the percentage of
mice developing convulsions with the standard and new insulin
preparation is compared.
Principle 4) The method should estimate, as far as possible & allow
an estimate of the error due to biological variation in different
animals/ persons at any one time, and in the same animal/ person
at different time. 7

8. Precautions to minimize error due to biological variation are-
• The experimental conditions must be kept constant,
• The biological response (indicator) should be sensitive to the
drug,
• The indicator should be insensitive to other drugs,
• The indicator should be capable of giving constant and
reproducible results,
• A preparation of known concentration (standard) must be
available to compare the response
• The number of experiments should be sufficiently large, and the
assay is designed to minimize biological variation, and
• The animal should be of the same species and strain (preferably
litter mates), of similar age, weight and sex, kept on similar diet,
and housed under similar conditions.
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Sources of error in bioassays-
Biological Variation and Selection of False Methodology

9. Standard preparation (Reference Standard)
A selected representative of the substance which serves as a
basis for the comparative measurement of activity.
These standard preparations are distributed to experts,
pharmacological firms and research laboratories to
standardize new batches of their preparations.
In India these reference standards are maintained and
distributed by the central drug research laboratories,
Calcutta, and the central research institute, Kasauli.
In USA, they are maintained by the national institute of
medical research, London.
The expert committee on the biological standardization of the
WHO also maintains and distributes these preparations to
various laboratories.
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10. Standard preparation (Reference Standard)
Use of standards
Biological assays are designed to measure relative potency of
two preparations, usually a standard and an unknown or
test.
Use of standard substance for comparison helps in solving the
problems arising from biological variations.
The observed response or effect of the unknown would be
always relative to the effect that produced by a standard
substance.
The standard substance is a pure substance, and in official
bioassays, it refers to pharmacopoeial standard.
In case of hormones, biological products and vaccines, it is
often necessary to establish the standard response of
standard substance against which unknown or test samples
can be calibrated. 10

11. Types of bioassays
Bioassays are of two types namely quantal response and
graded response bioassay.
Quantal response bioassay is ‘all or none’ phenomenon, e.g.
insulin induced hypoglycaemic convulsions in mice or
digitalis induced cardiac arrest in guinea pig. In both these
cases, the end point is all or none response that means
either convulsions or no convulsions, similarly either cardiac
arrest or no cardiac arrest.
Graded response bioassays are based on the observations that
there is a proportionate increase in the observed response
with a subsequent increase in the concentration or dose. It
measures the nature of the effect that substance is
expected to produce, e.g. contraction of smooth muscle
preparation for assaying histamine or the study of blood
pressure response in case of adrenaline. 11

12. Types of graded response bioassay
a) Matching bioassay
It is the simplest type of bioassay.
In this type of bioassay, the response of the test substance is
taken first and the observed response is tried tom match
with the response that is obtained with the standard drug.
Several responses of the standard drug are recorded till a
close matching response to that of test substance is
observed. A corresponding concentration is thus calculated.
This assay is employed when the sample size is very small.
Since the assay does not involve the recording of concentration
response curve (CRC), the sensitivity of the preparation is
not taken into consideration. Therefore, the precision and
reliability of such bioassay is not good.
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13. Types of graded response bioassay
b) Interpolation bioassay
In this type of bioassay, a concentration response curve (CRC)
or dose response curve (DRC) of a standard substance is
first established. Then record 2-3 responses due to test
substance. The selection of test responses should be such
that they lie on the linear portion of the concentration
response curve (CRC) of the standard drug.
The precision and reliability of this bioassay is much better as
compared to matching bioassay as the sensitivity of
preparation is assessed prior to testing the unknown or test
substance.
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14. Types of graded response bioassay
c) Bracketing bioassay
This method is made use of when the test sample is small.
It is also a simple assay procedure.
The response due to test substance is bracketed between two
responses (greater and smaller) of a standard substance.
The precision and reliability of this bioassay is also poor as the
sensitivity of the preparation is not taken into consideration.
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15. Types of graded response bioassay
d) Multiple point bioassays
 Three point bioassay
In such type, two responses of standard drug and one response due
to test sample are taken into consideration. The test response
should be intermediate between two responses of standard drug.
 Four point bioassay
In such type, two responses of standard drug and two responses of
test drug are recorded. The selection of two responses of
standard should be such that they lie on the linear portion of the
concentration response curve and also the ratio between the
doses should be 1:2. The selection of test response is determined
by hit and trial method so that the responses fall on the linear part
of the curve.
The precision, reliability and reproducibility of this bioassay are very
high. It is most commonly used for estimating the concentration
of the unknown sample. 15

16. Types of graded response bioassay
d) Multiple point bioassays
 Six point bioassay
In such type, three concentrations of standard drug and three
concentrations of test substances are used. The selection of
doses is done as described in four point bioassay. It is very time
consuming procedure.
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17. Latin square designs-
Multiple point bioassays
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S1 S2 T
S2 T S1
T S1 S2
S1 S2 T1 T2
S2 T1 T2 S1
T1 T2 S1 S2
T2 S1 S2 T1
S1 S2 S3 T1 T2 T3
S2 S3 T1 T2 T3 S1
S3 T1 T2 T3 S1 S2
T1 T2 T3 S1 S2 S3
T2 T3 S1 S2 S3 T1
T3 S1 S2 S3 T1 T2
Three point bioassay
Four point bioassay
Six point bioassay

18. Intact animal studies
Some simple experiments on intact animals have been described
which will help the students to understand and appreciate the
action of drugs.
These experiments are particularly designed so because the
pharmacy students has little accessibility to human patients
although they studies in detail the pharmacology of individual
drugs, e.g. study of routes of drug administration, onset of action,
study of cateleptogenic effect (extrapyramidal side effects) of
drugs, study of various types of convulsions, study of muscle
relaxing property, study of pain, analgesia and inflammation are
some of the procedures described.
If a good coordination is kept between the theory taught in the class
and the relevant practical demonstrated or done in the
subsequent practical turn, it will not only help the students to
understand the subject matter better but also make the study of
subject more interesting as well.
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Thank you!