UNIT 2.1 Enzyme-Linked Immunosorbent Assays

  1. Peter Hornbeck

Published Online: 1 MAY 2001

DOI: 10.1002/0471142735.im0201s01

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Hornbeck, P. 2001. Enzyme-Linked Immunosorbent Assays. Current Protocols in Immunology. 1:I:2.1:2.1.1–2.1.22.

Author Information

  1. University of Maryland, Baltimore, Maryland

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAR 1992

This is not the most recent version of the article. View current version (3 AUG 2015)


This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. The Support Protocol can be used to optimize the different ELISAs. The second support protocol presents a method for preparing alkaline phosphatase conjugates.