ELISA Virtual Lab

ELISA Sandwich: Theoretical Background

ELISA begins with a coating step, where the first layer either an antigen or an antibody is absorbed into a polystyrene 96 well plate. The coating is followed by blocking and detection steps and finally reading the results on the  ELISA reader.

There are four basic ELISA formats, allowing for a certain amount of flexibility which can be adjusted based on the antibodies available, the results required, or the complexity of the samples:

1. Direct ELISA.
2. Indirect ELISA.
3. Sandwich ELISA.
4. Competition or Inhibition ELISA.

ELISA is one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng.

The ELISA simulation lab begins with a coating step, where the first layer—either an antigen or an antibody—is absorbed into a polystyrene 96-well plate. This coating is followed by blocking and detection steps, and finally reading the results on the ELISA reader. This immunoassay exploits the antigen-antibody reaction to specifically detect analytes in complex samples.

There are four basic ELISA formats, allowing for a certain amount of flexibility which can be adjusted based on the antibodies available, the results required, or the complexity of the samples:

• Direct ELISA
• Indirect ELISA
• Sandwich ELISA (used here)

Competition or Inhibition ELISA
The Sandwich ELISA is one of the most sensitive immunoassays available.

The ELISA Sandwich Principle of Work

The enzyme linked immunosorbent assay (ELISA) is a common laboratory technique used to quantitatively measure the concentration of an analyte (usually antibodies or antigens) in solutions by detecting antigen-antibody reactions.

• The ELISA steps result in a colored end product, which correlates with the amount of analyte present in the original sample.
• In this ELISA virtual lab, we perform the sandwich ELISA to detect the presence of a specific antigen.
• Sandwich ELISA typically uses matched antibody pairs, where each antibody binds to different, non-overlapping parts of the antigen molecule. The first antibody, known as the capture antibody, is coated onto the plate. Next, the analyte or sample solution is added to the well to allow antigen binding.
• A second antibody layer, the detection antibody, is added to measure the analyte concentration. If the detection antibody is conjugated to an enzyme, this assay is called a direct sandwich ELISA. If unlabeled, a secondary detection antibody is used, resulting in an indirect sandwich ELISA.