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Immunology · Immunology lab

ELISA, western blot and rapid tests !

  • Topic

ELISA uses an enzyme that makes colour when an antibody binds its target. It underlies many screening tests, such as HIV tests.

Updated 2026-11-01

What it is

ELISA (enzyme-linked immunosorbent assay) fixes an antigen or antibody to a surface, lets the sample react, and reveals binding with an enzyme-labelled antibody that turns a substrate coloured; the colour is proportional to what is being measured. Western blot separates proteins by size and shows, band by band, which ones the serum has antibodies against. Rapid tests apply the same principle on a lateral-flow strip.

Key facts

  • Direct or capture ELISA detects antigen; indirect ELISA detects antibodies; in competitive ELISA more analyte gives less colour.
  • ELISA is very sensitive, ideal for screening; its positive predictive value falls when prevalence is low.
  • Western blot: electrophoresis of viral proteins, transfer to a membrane and detection of antibodies to each band (p24, gp41, gp120/160 in HIV).
  • Fourth-generation HIV tests detect antibodies and the p24 antigen, which shortens the window period.

Step by step

  1. Indirect ELISA: the well is coated with the microbe's antigen.
  2. Serum is added; specific antibodies, if present, bind the antigen.
  3. A wash removes what did not bind.
  4. An enzyme-labelled anti-human antibody is added.
  5. Another wash, then the substrate: the enzyme produces colour.
  6. A reader measures optical density and compares it with a cut-off.

In the lab

  • Current laboratory HIV algorithm (CDC): 1) antigen-antibody immunoassay; 2) if reactive, an immunoassay that differentiates HIV-1 from HIV-2; 3) if that is negative or indeterminate, a nucleic acid test (qualitative viral RNA).
  • Positive nucleic acid test with negative differentiation = acute HIV-1 infection.
  • Western blot was dropped from the CDC algorithm: it is less sensitive in early infection and handles HIV-2 poorly.
  • Where there is no laboratory, diagnosis often uses two or three different rapid tests in sequence, following the national strategy.
  • Approximate window: antibodies 23 to 90 days; laboratory antigen-antibody 18 to 45 days; nucleic acid 10 to 33 days.

Test yourself

Think of the answer, then tap the question to see it.

What do fourth-generation HIV tests detect?

Antibodies to HIV-1 and HIV-2 plus the p24 antigen, so they detect infection earlier than antibody-only tests.

In the current laboratory algorithm, what follows a reactive initial immunoassay with a negative differentiation test?

An HIV-1 nucleic acid test; if positive, it indicates acute infection.

What does a western blot show?

Which proteins, separated by size on a membrane, the serum has antibodies against; each protein shows up as a band.

Why does a positive ELISA in a low-prevalence population need confirmation?

Even with high specificity, at low prevalence a large share of positives are false positives (low positive predictive value).

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Educational content on the immune system; not a treatment or individual vaccination guide and no substitute for a health professional's advice. No doses are given.

Sources: Suggested Reporting Language for the HIV Laboratory Diagnostic Testing Algorithm (Centers for Disease Control and Prevention (CDC) and APHL, 2013); HIV Screening Test (MedlinePlus (U.S. National Library of Medicine), 2024); ENZYME LINKED IMMUNOSORBENT ASSAYS REVISITED (Medical Journal Armed Forces India (PubMed Central), 1997); Latex agglutination test (MedlinePlus (U.S. National Library of Medicine), 2024).

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