Direct ELISA Test for Antigen Detection

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.