Direct ELISA (Sandwich ELISA) Test for Antigen Detection

Enzyme-linked immunosorbent assay (ELISA) is extremely sensitive test that is used to detect specific antibodies or antigens. The direct ELISA is a test for the detection of antigen using specific immobilized antibody.

In this procedure, a known antibody is adsorbed to the inside of the well in a microtiter plate. After rinsing to remove excess antibodies, the sample suspected of containing antigen is added.

Next, an enzyme-linked antibody that can react with the antigen is added. If antigen is present in the well, the enzyme-linked antibody binds to it and is retained.

The colorless substrate for the enzyme is then added. Development for color (after enzyme-substrate reaction) indicates the presence of the antigen.

Outline of Direct ELISA Procedure and Mechanism

  1. Antigen specific antibody (Ab1) is attached to a solid phase surface.
    (i.e. Antibody directed against specific infectious agent in question is firmly fixed to a solid matrix, either the inside of the walls of microdilution tray or the outside of a spherical plastic or metal bead or some other solid matrix)
  2. Test specimen is added
    if antigen is present in the fluid to be tested, stable antigen-antibody complexes is formed; unbound antigen is thoroughly removed by washing.
  3. Second antibody against the antigen being sought is then added to the system. This antibody has been complexed to an enzyme such as alkaline phosphatase or horseradish peroxidase.
  4. If the antigen is present on the solid matrix, it binds the second antibody, forming a sandwich with antigen in the middle. Unbound labeled antibody is removed by washing.
  5. A chromogenic enzyme substrate is added. The hydrolysis of the enzyme substrate causes the color change and completes the action.
  6. The color developed is proportional to the amount of antigen present in the test specimen.

Results and Interpretation 

The intensity of the color is directly proportional to the amount of the antigen present in the same.  Amount of antigen present in the test sample can be measured using the ELISA reader.