What is measured by Sandwich ELISA?

Sourav PanSeptember 4, 2024

Answered

Sandwich ELISA (Enzyme-Linked Immunosorbent Assay) is a technique used to measure the concentration of specific antigens in a sample. The key features and steps of Sandwich ELISA are:

1. Coating with Capture Antibody:
   • The assay begins by coating a solid surface (usually a microtiter plate) with a specific capture antibody that binds to the target antigen. This antibody is designed to recognize and bind specifically to the antigen of interest.
2. Blocking:
   • After coating, a blocking step is performed to prevent non-specific binding of proteins to the plate surface. This is usually done with a blocking buffer containing proteins or other substances that bind to the surface, blocking any remaining unoccupied sites.
3. Binding of Antigen:
   • The sample containing the target antigen is added to the plate. If the antigen is present, it will bind to the capture antibody on the plate.
4. Detection with Secondary Antibody:
   • After washing away unbound materials, a secondary antibody, which is specific for the target antigen and is conjugated to an enzyme (such as horseradish peroxidase or alkaline phosphatase), is added. This antibody binds to the antigen that is captured on the plate.
5. Substrate Addition and Detection:
   • A substrate for the enzyme is added. The enzyme-substrate reaction produces a measurable signal, typically a color change. The intensity of the color is proportional to the amount of antigen present in the sample.
6. Quantification:
   • The color intensity is measured using a spectrophotometer. The resulting absorbance readings are compared to a standard curve to determine the concentration of the antigen in the sample.