The ELISA Immunoassay (IND-3)

The ELISA (enzyme-linked immunosorbant assay) is a powerful immunological method for detecting specific proteins in complex protein mixtures. The ELISA has become an important tool for the cell and molecular biologist. It is increasingly being applied in clinical medicine for detecting proteins associated with disease including antibodies produced in response to infection by the HIV virus. The method is also easy to perform and yields graphic results making it well suited for the teaching laboratory. In this exercise, students carry out the ELISA to study the specificity of antibody-antigen interactions. The basic procedures and typical results of the exercise are shown on the right. The 2-3 hour exercise was designed for sixteen students working in pairs and includes: microtitration plates, transfer pipets, chicken serum, cow serum, horse serum, rabbit serum, gelatin, tris buffer saline, tris buffer saline + NP-40, goat anti-rabbit IgG-peroxidase, TMB-peroxidase substrate, and hydrogen peroxide. Microliter dispensers are needed but not provided, and a colorimeter is desirable but not absolutely necessary.

Serum Concentration (Left to Right)

1. Varying dilutions of serum proteins from chicken (D and H), cow (C and G), horse (B and F), and rabbit (A and E) are adsorbed to the surface of wells of a microtitration plate. The antigen used for the experiment is the IgG molecule from rabbit serum.

2. The immobilized serum proteins are incubated with a peroxidase-linked antibody to the rabbit antigen. During this incubation, the antibody binds preferentially to the antigen in rabbit serum.

3. The bound antibody is detected by a color-producing reaction and the amount of color development is proportional to the amount of antigen present. Note maximal color development occurs with the rabbit serum proteins.