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Paper I — 2020 February (Supplementary) (2010 Scheme) — Q8

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Paper I
2020 February (Supplementary) (2010 Scheme) · 40 marks · 120 min

Question

Immunofluorescence.

Q82 marksShort Notes

Answer

Immunofluorescence is a laboratory technique that uses fluorescent-dye-labelled antibodies to detect and visualize a specific antigen (or antibody) in a sample under a fluorescence microscope, exploiting the specificity of antigen-antibody binding combined with the visual sensitivity of fluorescence.

Principle: a fluorochrome (fluorescent dye, e.g., fluorescein isothiocyanate, FITC — emits green fluorescence; or rhodamine — emits red) is chemically conjugated to an antibody without destroying its antigen-binding specificity; when this labelled antibody binds its target antigen in a sample, the resulting complex fluoresces (glows) under ultraviolet/blue light exposure in a fluorescence microscope, allowing direct visualization and localization of the antigen.

Types:

  • Direct immunofluorescence (DIF) — a fluorochrome-labelled antibody is applied directly to the sample and binds its specific antigen in a single step; simple and rapid, but requires a separately labelled antibody for each different target antigen, limiting flexibility.
  • Indirect immunofluorescence (IIF) — an unlabelled primary antibody (specific for the target antigen) is applied first, followed by a fluorochrome-labelled secondary antibody (directed against the species/class of the primary antibody, e.g., anti-human IgG); this amplifies the signal (multiple labelled secondary antibodies can bind each primary antibody, increasing sensitivity) and allows the same labelled secondary antibody to be used with many different unlabelled primary antibodies, making it more versatile and widely used.

Applications:

  • Rapid identification of microorganisms directly in clinical specimens (e.g., direct fluorescent antibody test for rabies virus antigen in brain tissue, Chlamydia trachomatis in genital/conjunctival smears, Bordetella pertussis in nasopharyngeal secretions, Legionella in respiratory specimens).
  • Detection of circulating autoantibodies (e.g., antinuclear antibody testing in SLE, using patient serum as the “primary antibody” against a fixed cell substrate, detected by fluorochrome-labelled anti-human immunoglobulin — an indirect immunofluorescence format).
  • Confirmatory serological testing (e.g., FTA-ABS test for syphilis).
  • Immunohistochemistry/immunopathology, localizing immune complex deposition in tissue biopsies (e.g., renal biopsy in glomerulonephritis).
  1. 1General Properties of Viruses and Virus Host Interaction

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