U01.01.040 Enzyme-linked immunosorbent assay

The Enzyme-Linked Immunosorbent Assay (ELISA) is an immunologic test used to detect either a specific antigen or antibody in a patient’s blood sample. It relies on the interaction between an antibody and an enzyme linked to it, producing a detectable signal upon reaction with a substrate. ELISA is commonly used for diagnostic purposes and research applications due to its high sensitivity and specificity.

Key Concepts

Purpose: Detect specific antigens or antibodies in blood.
Mechanism: Antibody linked to an enzyme; substrate reaction produces a detectable signal.
Sensitivity and Specificity: High, but less specific than Western blot.

Types of ELISA

Type	Description
Direct ELISA	Detects antigens directly using an enzyme-linked antibody.
Indirect ELISA	Detects antibodies against specific antigens.
Sandwich ELISA	Used for detecting antigens; involves two antibodies.
Competitive ELISA	Measures the amount of antigen by competition with a labeled antigen.

Steps Involved in ELISA

Coating: The antigen or antibody is coated onto a microplate.
Blocking: Non-specific sites are blocked to prevent false signals.
Detection: Antibody linked to an enzyme is added. If the antigen is present, the antibody binds to it.
Substrate Addition: Substrate is added, producing a detectable color change or signal.

Points to Remember

ELISA is highly sensitive and can detect very low concentrations of antigens or antibodies.
Western blot is more specific but less sensitive compared to ELISA.
ELISA is commonly used for HIV, hepatitis, and allergy testing.
False positives can occur in ELISA, but its sensitivity makes it a valuable first-line diagnostic tool.

Key Differences: ELISA vs. Western Blot

Aspect	ELISA	Western Blot
Purpose	Detects antigens or antibodies in blood.	Identifies specific proteins based on size and immunoreactivity.
Sensitivity	High sensitivity, good for initial screening.	Lower sensitivity but higher specificity.
Specificity	Lower than Western blot.	Higher specificity.
Common Use	First-line test for viral infections (e.g., HIV).	Confirmatory test after ELISA in case of indeterminate results.

Learning Objective

Understand the principles of ELISA, its types, and applications, and differentiate it from other diagnostic tests like Western blot.

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