Page 137 - MNUmicrobiology practical 2025

General Microbiology & Immunology (PM 401) second level Semester 4 2024/2025

ELISA

What Is ELISA?

• ELISA is the basic assay technique, known as enzyme-linked immunosorbent assay

(also referred to as EIA: Enzyme Immunoassay) that is carried out to detect and

measure antibodies, hormones, peptides and proteins in the blood.

• The method depends on conjugation of an enzyme to either Ag or Ab, then substrate is

added as a quantitative measure of enzyme activity.

Direct ELISA

• Used for direct detection of Ags.

• Known specific Ab is immobilized by adsorption onto a plastic surface.
• A clinical sample is added (if Ag present it will bind to the immobilized Ab)

• enzyme- linked Ab specific for the test antigen is added (attach to the fixed Ag if present)
• wash the excess

• add the substrate
• A substrate is a chemical which is converted by the enzyme into a color or fluorescent

or electrochemical signal.

• Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the

plate wells to determine the presence and quantity of antigen.

• Disadvantages: expensive method (for each Ag we need specific Ab labelled)

General Microbiology & Immunology (PM 401) Practical note Page 126