Principles of bioassay

1. Principle of Bioassay
Dr. S. Parasuraman, M.Pharm., Ph.D.,
Associate Professor & Unit Head,
Unit of Pharmacology,
Faculty of Pharmacy, AIMST University,
Bedong 08100, Malaysia.

2. Learning Outcomes
•Upon successful completion of this topic, students
should be able to:
• Discuss the importance and purpose of bioassay (CLO1)
• List criteria for bioassays (CLO 1)
• Highlight the errors in bioassay and their preventive
measures (CLO1)

3. Bioassay
• Bioassay comprises of “bio” means living material and
“assay” means assessment at laboratory, i.e., assessment of
unknown substance on any living tissue.
• Bioassay is defined as estimation or determination of
concentration or potency of physical, chemical or biological
agents by means of measuring and comparing the
magnitude of the response of the test with that of standard
over a suitable biological system under standard set of
conditions.

4. Substances Used in Bioassay
•Substances derived from plants and animal sources
(hormones, proteins) are mainly assessed by biological
assay.
•Chemical and synthetic drugs are ideally not required
for bioassay because of known structure. Moreover,
their own chemical standardization methods are
available.

5. Objective or Purpose of Bioassay
•Bioassays have mainly three main constituents namely,
stimulus, subject and response. Through these
constituents, one can make
• Identification of various compounds
• Quantify the screening procedure and
• Commercial production of drugs like antibiotics.

6. Objective or Purpose of Bioassay
•Bioassays have mainly three main constituents namely,
stimulus, subject and response. Through these
constituents, one can make
• Identification of various compounds
• Quantify the screening procedure and
• Commercial production of drugs like antibiotics.

7. Principle of Bioassay
•A bioassay is a quantitative procedure using a functional
response in a living system, either in vivo or in vitro.
•Bioassay involves the comparison of the main
pharmacological response of the unknown preparation
with that of the standard.
•A bioassay measures the ‘relative activity’ or ‘potency’
of a substance and its specific capacity to achieve an
intended biological effect.

8. Principle of Bioassay
•The method selected should be reliable, sensitive, and
reproducible and should minimize errors due to
biological variation and methodology.
•Bioassay might measure a different aspect of the same
substance compared to chemical assay. The test
solution and standard should be compared for their
established pharmacological effect using a specified
pharmacological technique.

9. Principle of Bioassay

10. Criteria of bioassay
• Critical attributes of bioassays are defined in ICH and USP guidelines
and include specificity, accuracy, precision, linearity, range and
robustness.
• Specificity: Specificity (concerned with the accuracy) is normally
evaluated as a part of bioassay development or validation.
• Accuracy: Accuracy is a measure of how close results are to a target
or reference value.
• Precision: Measure of repeatability of multiple assays of the same
homogeneous sample under specific condition.
• Linearity: Ability of your assay to provide measured values that are
proportional to the concentration of the sample.
• Range: The limits between which variation is possible.
• Robustness: Robustness is the property of being strong and healthy
in constitution.

11. Applications of bioassay
•According to several pharmacopeias, assay used for
• estimate potency of natural drug, for which chemical
method is not known or established.
• standardization of drugs of natural origin (plant and
animal origin) whose structure or origin is unpredictable
• screening of new compound for biological activity.
• estimation of biologically active substance like histamine,
acetylcholine, 5-hydroxytryptamine, adrenaline,
bradykinin, substance P, prostaglandins, etc.
• estimation of ED50/TD50 and LD50. (presently instead of
LD50, NOAEL or LD10 is preferred).

12. Errors in bioassay
•While performing bioassay, sometime responses are
varied (increase/ decrease), which is calculated as error
of the procedure.
•Broadly error is divided into two categories, i.e., due to
biological variation or due to methodological error.

13. Errors in bioassay - biological variation
• Animal to animal or individual biological variation occurs in
response to a drug action- it is known. The effect of drug
dose to the tissue response may vary and will fall in a range.
• Reason and its correction for biological variation
• Downregulation of receptors (avoid over washing)
• Loss of tissue sensitivity (change the tissue)
• Laboratory condition may be variable (must use same species)
• Housing and handling of animals (must be handled by a
qualified experienced staff)

14. Errors in bioassay - methodological error
• Usually, this error occurs due to the faulty method selection
or due to the incorrect procedure, i.e., human error and
experimental error.
• Reasons for Methodological error
• Lack of standardization of procedure
• Set-up of apparatus
• Tissue isolation/extraction and preparation for the experiment
• Preparation of physiological salt solution and maintenance of its pH
• Drug preparation or it dilution