Principles of bioassay

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 ED 50 /TD 50 and LD 50 . (presently instead of LD 50 , NOAEL or LD 10 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