BIOLOGICAL ASSAYS

Biological Assays

Includes the quantitative assay of drugs using intact animals,animal preparations, isolated living tissues and cells, ormicroorganisms

Bioassays

Are conducted by determining the amount of a preparation of unknown potency required to produce a definite effect on suitable test animals or organs under standard conditions.

- Similarities to humans (especially mammals: similar DNA & evolutionary traits)
- Adjustable living environment
- Shorter lifespan – allows us to observe effects across the entire life cycle

Why Use Animals in Bioassays?

Reduction
Refinement
Replacement

IACUC 3R’S

Reduction

IACUC 3R’S
Use the minimum number of animals needed to produce reliable results

Refinement

IACUC 3R’S
Follow rules/regulations approved by ethics committees

Replacement

IACUC 3R’S
Use in vitro or other alternatives if validated and standardized

Institutional Animal Care and Use Committee

- Reviews animal protocols
- Requires documentation
- Ensures ethical handling before, during, and after testing

1. If the chemical identity of the active principle has not been elucidated fully

2. If no adequate chemical assay has been devised for the active principle, although its chemical structure has been established (e.g.,insulin).

3. If the drug is composed of a complex mixture of substances of varying structure and activity (e.g. digitalis).

4. If purification of the crude drug, sufficient for the performance of a chemical assay, is not possible or practical(e.g., the separation of vitamin D from certain irradiated oils).

5. If the chemical assay is not a valid indication of biological activity, due, for example, to lack of differentiation between active and inactive isomers.

Biological standardization procedures generally are less precise,more time-consuming, and more expensive to conduct than arechemical assays; therefore, they generally are reserved for use:

Reference Standard

Has a known purity and would elicit a certain biologic effect.

Treatment Groups

Refers to the group (or sample) being tested, usually a drug or substance of unknown potency or new formulation. It is the experimental sample that you want to evaluate.

Insulin

If no adequate chemical assay has been devised for the active principle, although its chemical structure has been established

Digitalis

If the drug is composed of a complex mixture of substances of varying structure and activity

The separation of vitamin D from certain irradiated oils

If purification of the crude drug, sufficient for the performance of a chemical assay, is not possible or practical

Glucagon Assay using Cats

Is used in the treatment of acute hypoglycemic reactions, especially in diabetics with insulin-induced hypoglycemia.

Elevation of Blood Glucose

Glucagon Assay using Cats/ Rat Hepatocytes
Endpoint of Testing:

Glucagon Assay using Rat Hepatocytes

An ex vivo procedure in which the drug’s effects are assessed on a primary culture of rat liver cells.

75

What percent is used for Glucagon Assay using Rat Hepatocytes?

Microbiological Assays

- To determine the activity/potency of antibiotics.
- To facilitate in determination of the antibiotic appropriate for patient recovery

Cylinder Plate
Turbidimetric

Two Methods for Microbiological Assay

Plate Assay

Another Term for Cylinder Plate

Tube Assay

Another term for Turbidimetic

Cylinder Plate

- Diffusion of the antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish
- Circular area around the cylinder is observed (Zone of Inhibition)

Effective Antibiotic

CYLINDER PLATE
Bacterial concentration is reduced around the disc; zone of inhibition is visible.

Ineffective Antibiotic

CYLINDER PLATE
Bacterial Concentration does not change.

Very Effective Antibiotic

CYLINDER PLATE
Bacterial concentration is significantly reduced around the disc; a large zone of inhibition is visible.

Turbidimetric

Inhibition of the growth of a microbe in a uniform solution of the antibiotic in a fluid medium that is favorable to the growth of the microbe in the absence of the antibiotic

Spectrophotometer

Instead of ZOI, in turbidimetric what is used to measure the absorbance?

Decrease Potency (bacteria grew more)

TURBIDEMITRIC
Increase Turbidity

High potency (growth inhibited)

TURBIDEMITRIC
Decrease Turbidity