Immunoassay

Core Principles of Immunoassay

  1. Antigen-Antibody Binding:
    • The fundamental concept behind immunoassays is the highly specific binding between an antibody and an antigen.
    • Antibodies recognize and bind to unique molecular structures on antigens (epitopes).
  2. Specificity and Sensitivity:
    • Specificity: The ability of the antibody to bind only to the target antigen.
    • Sensitivity: The ability to detect very small amounts of the target substance.
  3. Labeling and Detection:
    • To measure the binding, either the antibody or antigen is labeled with a detectable marker:
      • Radioisotopes (Radioimmunoassay, RIA)
      • Enzymes (Enzyme-linked immunosorbent assay, ELISA)
      • Fluorescent tags (Fluoroimmunoassay)
      • Chemiluminescent agents
    • The intensity of the signal correlates with the amount of antigen or antibody in the sample.

Types of Immunoassays

  1. Competitive Immunoassay:
    • A labeled antigen competes with the sample antigen for binding to an antibody.
    • More sample antigen → less labeled antigen binds → inverse signal.
  2. Non-Competitive (Sandwich) Immunoassay:
    • The antigen is “sandwiched” between two antibodies: a capture antibody and a labeled detection antibody.
    • More sample antigen → more sandwich formation → direct signal.
  3. Direct vs Indirect Immunoassay:
    • Direct: Uses a labeled primary antibody.
    • Indirect: Uses an unlabeled primary antibody and a labeled secondary antibody for amplification.

Common Immunoassay Formats

  • ELISA: Most common; uses enzyme labels to produce a colorimetric or luminescent signal.
  • Western blot: Combines immunoassay with protein separation.
  • Lateral flow assays: Used in pregnancy tests and rapid antigen tests.
  • Radioimmunoassay (RIA): Uses radioactive labels; very sensitive but less commonly used due to safety concerns.

Applications

  • Clinical diagnostics: Detect hormones, drugs, pathogens, or antibodies (e.g., HIV, COVID-19).
  • Research: Quantify proteins, cytokines, etc.
  • Pharmacokinetics: Monitor drug levels in the body.

Key Concepts in Performance

  • Affinity and Avidity: Strength of antibody-antigen interaction.
  • Hook effect: Very high antigen levels can falsely reduce signal in sandwich assays.
  • Cross-reactivity: Non-specific binding can affect assay accuracy.