Western blot
Summary
Western blot is a laboratory technique used to detect specific proteins in a sample by separating them based on molecular weight and identifying them with labeled antibodies. It is classically known as a confirmatory test for HIV infection after a positive ELISA screen, though HIV testing algorithms now favor other confirmatory assays.
Detail
Western blotting (immunoblotting) involves several steps: (1) protein extraction and denaturation, (2) separation by SDS-PAGE gel electrophoresis based on molecular weight, (3) transfer ('blotting') of proteins onto a nitrocellulose or PVDF membrane, (4) blocking non-specific binding sites, (5) incubation with a primary antibody specific to the target protein, and (6) detection using a labeled secondary antibody (enzyme-linked or fluorescent) that produces a visible band. The presence and position of bands indicate the identity and relative abundance of the protein.
Clinically, Western blot has classically been used as a confirmatory test following a positive ELISA/EIA screen for HIV antibodies, since ELISA has high sensitivity but Western blot offers higher specificity by detecting antibodies against specific viral proteins (e.g., gp41, gp120, p24). However, current CDC-recommended HIV testing algorithms now use a fourth-generation antigen/antibody combination immunoassay followed by an HIV-1/2 antibody differentiation immunoassay rather than Western blot, due to faster turnaround and improved detection during acute infection window periods.
Western blot is also used in diagnosing other conditions such as Lyme disease (as a two-tiered confirmatory test after positive ELISA) and in various research settings to study protein expression, post-translational modifications, and validate antibody specificity.
High-yield associations: ELISA (screening, high sensitivity) → Western blot (confirmatory, high specificity) is a classic testing paradigm tested on boards, particularly for HIV and Lyme disease diagnosis. Understanding the trade-off between sensitivity and specificity in sequential testing strategies is a key concept.
Sources
- First Aid for the USMLE Step 1
- Kaplan USMLE Step 1 Lecture Notes: Immunology
- CDC HIV Testing Algorithm Guidelines
- Harrison's Principles of Internal Medicine
Reviewed by AnkiBoss editorial — medical student review. Information here is for study reference only and is not medical advice. Spotted an error? Let us know.