ELISA test : Antibody Detection

he Enzyme Linked Immuno-sorbent Assay or ELISA is a commonly used format for serologic testing. ELISA serologies are usually done on multi-well microtiter plates so that dilution of serum are easily prepared and tested.

Procedure and Principle for Indirect Assay:
  1. Wells of the plate are coated with the antigen of interest
  2. Wells are filled with dilution of the patient's serum. If the antiboady (1st antibody) against the antigen are present in the serum, they will be immobilized due to binding to the antigen fixed to the bottom of the wells.
  3. Wells are then washed to remove all the unbound antibodies (1st antibodies).
  4. Then, a solution of animal antibody against the human antibody (2nd antibody) i.e. antihuman antibody or immunoglobulin covalently conugated (linked) with an enzyme.
  5. Wells are washed again to remove the unbound enzyme linked antihuman antibody (2nd antibody).
  6. Finally, a solution of colorigenic enzyme substrate is added.
  7. The interaction of the substrate with the enzyme on the 2nd antibody (antihuman antibody) generates visible color.
  8. Read results directly through the bottom of the microwell plate using an automated or semi-automated photometer (ELISA-reader).
Similarly, ELISA test can also be used to detect antigens in the specimen collected. The principle for antigen detection has been illustrated in the image below:

Some Common Uses of ELISA:

  1. Screening test for HIV
  2. Detecting potential food allergens
  3. HCG pregnancy test