Introduction to ELISA
Microbiology 542 -- Eric Martz


Enzyme-Linked ImmunoSorbent Assays (ELISA) are widely used in research, diagnosis, and testing because of they are affordable and sensitive to tiny amounts of material. Usually, the assay involves multiple binding and rinsing steps, followed by spectrophotometry. However, in some cases, properly engineered assays can be done in a single step giving qualitative results readable by eye. The pregnancy tests commonly available in drug stores use ELISA to detect the human chorionic gondotropin hormone in urine. For a great animation of how this works, go to, click on Animations, then Chapter 6 Antigen-Antibody Reactions. There you will find additional links to animations and tutorials on ELISA.

ELISA assays can be done in several different ways called "direct", "indirect", "sandwich", and "competition" ELISA. Consult your favorite textbook of immunology for details.

ELISA assays use antibodies that are covalently linked ("conjugated") to enzymes. Antigen is bound to a plastic well, and enzyme-linked antibody is bound to the antigen. Unbound antibody is rinsed away. The amount of enzyme remaining, and hence the amount of antibody bound, is reported by adding a substrate that changes color when acted upon by the enzyme. The absorbance of the resulting color is proportional to the amount of enzyme bound to the well, hence to the amount of antibody, hence to the amount of antigen. Thus, color reports antigen, quantitatively.

The sensitivity of ELISA results from the amplification by enzyme activity. Each bound enzyme molecule can generate thousands of colored product molecules by enzymatic activity. Prior to enzyme-linked antibodies becoming widely available, radioactive antibodies were used in Radio-Immuno-Assays (RIA). The RIA was such a significant breakthrough that its inventor, Rosalyn Yalow, shared the 1977 Nobel Prize for Physiology and Medicine (


You have purified rabbit IgG through a series of steps, each hopefully resulting in increasing purity. The amounts of contaminents in each step could be visualized qualitatively in your SDS-PAGE results. You have quantitated total protein in each stage with A280. ELISA is capable of quantitating the concentration of rabbit IgG in each stage. The purity, or percentage of the protein in each sample that is rabbit IgG, can then be calculated.