Enzyme-Linked Immunosorbent Assay (ELISA) | Zoology Optional Notes for UPSC PDF Download

ELISA Principles

Variations of ELISA:

• Direct ELISA: In this method, a primary antibody labeled with a reporter enzyme or tag directly interacts with the antigen. This approach can be applied when the antigen is immobilized on the assay plate or as part of the capture assay format. While less common in ELISA, it is frequently used for immunohistochemical staining of tissues and cells.

• Indirect ELISA: This variation is employed to detect antibodies or determine their concentration. A sample, containing the primary antibody (Ab1), is introduced to an antigen-coated microtiter well. After removing unbound Ab1, an enzyme-conjugated secondary antibody (Ab2) is added, which binds to Ab1. Subsequently, any unbound Ab2 is washed away, and a substrate for the enzyme is introduced. The amount of colored, fluorescent, or luminescent reaction product generated is measured using a specialized plate reader and compared to a standard curve with known Ab1 concentrations. This method is often used in ELISA to detect antibody presence. (A direct ELISA would detect the amount of antigen on the plate using enzyme-coupled antibodies, but it is rarely used.)

  

• Principle: In a sandwich ELISA, the antibody is immobilized on a microtiter well, rather than the antigen. A sample containing unknown antigen amounts interacts with the immobilized antibody. After washing to remove unbound components, a second enzyme-linked antibody, specific for a different epitope on the antigen, is added and allowed to bind to the captured antigen. Subsequently, after washing away unbound second antibody, a substrate is introduced, and the resulting colored reaction product is measured.

• Applications: Sandwich ELISAs are particularly useful for measuring soluble cytokine concentrations in tissue culture supernatants, serum, and body fluids.

• Principle: The competitive ELISA offers high sensitivity for measuring antigen amounts. In this method, an antibody is initially incubated in solution with a sample containing the antigen. The antigen-antibody mixture is then added to an antigen-coated microtiter well. A higher initial concentration of antigen in the sample decreases the amount of free antibody available to bind to the antigen-coated well. After removing unbound antibody, an enzyme-conjugated Ab2 specific to the Ab1 isotype can be introduced to assess the amount of bound Ab1. In the competitive assay, a higher antigen concentration in the original sample results in a lower final signal.

Applications of ELISA

• Serum Antibody Concentrations
• Detection of Potential Food Allergens: Milk, peanuts, walnuts, almonds, eggs, etc.
• Disease Outbreak Monitoring: Used to track the spread of diseases like HIV, bird flu, common colds, cholera, and STDs.
• Detection of Antigens: For example, pregnancy hormones, drug allergens, GMOs, and diseases like mad cow disease.
• Detection of Antibodies in Blood Samples for Past Exposure to Disease: Such as Lyme disease, trichinosis, HIV, bird flu, and more.