Techniques used in the assay of drugs (biopharmaceuticals)

1. Techniques used in assay of drugs

2. “An assay is an analytical measurement procedure that produces a
detectable signal for quantifying a biological process”
• Assay quality parameters: absorbance, fluorescence, luminescence, radioactivity, etc.

3. Characteristics of a good assay method
• Sensitivity
• Specificity
• Repeatability/Reproducibility
• Validity
• Stability

4. Types of assays
Biological assay
• These methods are performed on living animals, isolated living organ and tissue, or micro-organisms.
• The specific potency of drug is given to animal or human volunteers and then drug response is
compared with the standards.
Immunoassay
An immunoassay is an analytical method which uses antibodies as reagents to quantitate or detect
specific analytes.
Chemical assay: A chemical assay refers to the analysis of a sample material using a set of chemical
procedures.

5. Chemical assays
• A chemical assay refers to the analysis of a sample material using a set of chemical procedures
• Qualitative- assay methods that determine physicochemical properties
• Quantitative- volume or weight of the substance
• Chemical assays are more precise.
• They are less time-consuming.
• Moreover, they are less expensive and easy to handle, and less manpower is required.
• In chemical assays, active constituent and structure are fully known.

6. Chemical assay
Analytes measured/quantified using
• Photometry
• Colorimetry
• Spectrophotometry
• Fluorimetry
• Chromatography

7. Photometry
• When light is passed through a coloured solution, certain wavelengths are selectively absorbed giving a plot of the
absorption spectrum of the compound in solution.
• According to Beer-lamberts law the amount of light absorbed is proportional to the solute concentration present in
solution.
• ISO (International organization for standardization) recognises the importance of photometry for drug discovery
applications
• Absorbance (A)= log1/T

8. Chromatography
• Chromatography is a laboratory technique for the separation of a compound from a mixture. The mixture is dissolved in a fluid (gas, solvent, water, ...) called
the mobile phase, which carries it through a system (a column, a capillary tube, a plate, or a sheet) on which is fixed a material called the stationary phase. The
different constituents of the mixture have different affinities for the stationary phase. The different molecules stay longer or shorter on the stationary phase,
depending on their interactions with its surface sites.
• Differential affinities of the various components of the analyte towards the stationary and mobile phase results in the differential separation of the components.
• Mobile phase or carrier solvent moving through the column Stationary phase or adsorbent substance that stays fixed inside the column Eluent fluid entering the
column Eluate fluid exiting the column (that is collected in flasks)
• Elution: the process of washing out a compound through a column using a suitable solvent
Column chromatography: It separates substances based on differential adsorption of compounds
Paper chromatography: Paper chromatography works majorly on capillary attractions.
Thin layer chromatography: Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer
of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose.
Gas chromatography: Gas chromatography is a chromatography technique that can separate and analyze volatile compounds in gas phase.
High performance liquid chromatography : High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in
analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture
through a column filled with a solid adsorbent material.

9. Bioassay
• Bioassays utilize living cells or tissues to determine the activity of a substance
• 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
Principles of bioassay
• 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

11. Biological assay of drugs
• Biological assays are reproducible, reliable, and robust and biologically relevant.
1. Anti-inflammatory activity of drugs
2. Antipyretic activity of drugs
3. Anti-diabetic activity of drugs
4. Analgesic activity of drugs
5. Antiulcer activity of drugs
6. Anthelmintic activity of drug: they are performed on earth-worms
7. Cardiac activity of drug: the cardiac glycosides are evaluated on frog and pigeon
8. Microbiological methods: the living bacteria, yeast and molds are used for assaying vitamins and to determine
the activity of antibiotic drugs.

12. Biochemical versus Cell-based assays
Biochemical assays:
-Simple
-More consistency
-Direct measurement of target engagement
-Increased specificity of compounds
Disadvantages:
-May be non-physiological
-Not possible to determine compound
properties such as membrane permeability,
toxicity, off-target effect
Cell based assays:
More physiological, amenable to systems approach
-Can simultaneously assay for compound properties
(membrane permeability, toxicity, off-target effects
Disadvantages:
-Complex
-High rate of noise
-Exclusion of less soluble/permeable compounds

13. Immunoassay
• An immunoassay is an analytical method which uses antibodies as
reagents to quantitate or detect specific analytes
• Principle: Immunoassay uses antibody and antigen complexes as a
means of generating measurable results
• ELISA • Radioimmunoassay • Fluoroimmunoassay

14. ELISA
• ELISA detects the presence of an antibody in a sample
• The antigen is attached to the wells of the microtitre plate.
• A sample containing the antibodies is added to the antigen-coated wells for binding with the
antigen.
• The free primary antibodies are washed away and the antigen-antibody complex is detected
by adding a secondary antibody conjugated with an enzyme that can bind with the
primary antibody.
• All the free secondary antibodies are washed away. A specific substrate is added which gives
a coloured product.
• The absorbance of the coloured product is measured by spectrophotometry.

15. Radioimmuno assay
• Radioimmunoassay’s high sensitivity is based on these principles – strong binding reaction consists of
antigen vs antibody reaction. Its specificity is based on competitive binding reaction and radio
emission.
• A specific quantity of antigen is made radioactive.
• The radiolabeled antigen is mixed with a specified amount of antibody for a particular antigen causing
the two to bind to each other.
• A serum sample from a patient that contains an unknown quantity of the same antigen is added
causing the unlabeled antigen from the serum to compete with the radiolabeled antigen for the
antibody binding sites.
• The unlabeled antigen concentration increases causing it to bind more to the antibody.
• The radiolabeled variant is displaced reducing the ratio of antibody-bound radiolabeled antigen.
• The bound antigen separates from the unbound ones.
• The remaining free antigen’s radioactivity in the supernatant is measured

16. High Content Screening
• Facilitates validation of effects of compounds at the cellular and subcellular level (expression, localization,
morphological changes in cells.
• Macromolecules (e.g. proteins, RNA) are labeled with fluorescent tags
• Technology uses automated digital microscopy, flow cytometry and IT- systems for analysis and storage of data
• Slower than HTS
• Popular as secondary screens in drug
discovery programs