Unit

UNIT 11.2 Enzyme-Linked Immunosorbent Assays (ELISA)

  1. Peter Hornbeck1,
  2. Scott E. Winston (bacterial cell lysate antigens)2,
  3. Steven A. Fuller (bacterial cell lysate antigens)3

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb1102s15

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Hornbeck, P., Winston, S. E. and Fuller, S. A. 2001. Enzyme-Linked Immunosorbent Assays (ELISA). Current Protocols in Molecular Biology. 15:I:11.2:11.2.1–11.2.22.

Author Information

  1. 1

    University of Maryland, Baltimore, Maryland

  2. 2

    Univax Biologics, Rockville, Maryland

  3. 3

    Allelix Inc., Mississauga, Ontario

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JUL 1991

Abstract

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. Support protocols are provided for optimizing the different ELISAs and preparing lysates for use as test antigen from bacterial cultures containing expressed protein.