Enzyme Linked Immunosorbent Assay

Aka: Enzyme Linked Immunosorbent Assay, ELISA

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II. Mechanism

  1. Enzyme-linked antibodies, specific to a another Antibody or Antigen, are embedded in specific positions on a surface
    1. Surface is typically a labeled grid of wells
  2. Test solution containing a patient's serum or other body fluid is poured over the surface (immunosorbent substrate)
  3. The surface embedded antibodies on binding Antigens, activate enzymes resulting in a color change detected by machine
    1. Enzymes include horseradish peroxidase
    2. Degree of color change is proportional to the Antigen concentration in the test solution
    3. Machine detection is by spectrophotometry

III. Types

  1. Direct ELISA
    1. Standard ELISA method with single detection phase as described above
  2. Sandwich ELISA
    1. First ELISA phase binds non-specific Antigen (e.g. antibodies such as IgE)
    2. Second ELISA use the bound Antigen with additional markers to sub-classify the Antigen types
  3. Competitive ELISA
    1. ELISA method may use enzyme-linked Antigen instead of Antibody
    2. In first phase, a solution of Antibody binds to Antigen
    3. In next phase, another Antibody solution binds competitively to unbound or less specifically bound Antigen
  4. Reverse ELISA
    1. Antibodies and Antigens are incubated together
    2. Incubated fluid is moved to a scavenger container to remove unbound Antibody
    3. Remaining Antibody and Antigen complexes are measured via flow cytometry

IV. Labs: Examples

  1. HIV ELISA
  2. H. pylori ELISA
  3. Lyme ELISA
  4. RadioAllergoSorbent Test (RAST)
    1. Test for IgE Antibody related to Type 1 Hypersensitivity Reactions
    2. Performed as ELISA or with radiolabeled anti-IgE Antibody

V. Resources

  1. ELISA (Wikipedia)
    1. https://en.wikipedia.org/wiki/ELISA

VI. References

  1. Mahmoudi (2014) Immunology Made Ridiculously Simple, MedMaster, Miami, FL

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