Basic Protein and Peptide Protocols

Volume 32 of the series Methods in Molecular Biology™ pp 461-466

Enzyme-Linked Immunosorbent Assay (ELISA)

  • Denis J. ReenAffiliated withThe Children’s Research Centre, Our Lady’s Hospital for Sick Children

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The ELISA technique (1) is a simple, sensitive, rapid, reliable, and versatile assay system for the quantitation of antigens and antibodies. Because of the extreme discriminating power of antibodies to recognize an almost infinite array of antigenic structures, the application of ELISA to analyte measurement is almost unlimited. ELISAs have been developed in many configurations depending on the particular application of the assay. In practice, ELISA as a solid-phase technique may be classified into two main types: (1) competitive assays using either an antigen-enzyme conjugate or an antibody-enzyme conjugate, and (2) noncompetitive assays using a double antibody “sandwich” technique where the second antibody has an indicator enzyme conjugated to it. In solid-phase ELISA, one of the immunoreactants (antibody or antigen) is immobilized onto a solid support (microtiter plate) by adsorption, through noncovalent interactions. The immobilized antibody is then incubated with test solution containing the analyte of interest. Following a period of incubation and washing, the bound antigen is detected, by the addition of an enzyme-conjugated antibody that binds to the remaining antigenic sites on the antigen.