Enzyme-linked immunosorbent assay (ELISA)

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Principle of indirect ELISA

Principle of indirect ELISA

Term: 2014
Published: February 27, 2014
Revised: April 8, 2014

Enzyme-linked immunosorbent assays (ELISAs) are the most commonly used serological diagnostic tests. The OIE recommends ELISA as the prescribed or alternative test for international trade for most diseases. ELISAs are also available for a wide variety of other diseases and analytical assays. The three most commonly used variations of the ELISA are the indirect ELISAs; competitive and blocking ELISAs and antigen capture ELISAs.

In the indirect ELISA antigen is immobilised by coating it onto an ELISA microtitre plate. Serum suspected of containing antibody is then added and antibody, if present, attaches to the immobilised antigen and a second antibody (Ab2) that has been conjugated to an enzyme is used to identify the attached antibody.

Competitive and blocking ELISAs work on the principle that two antibodies compete for a binding site on an epitope. Competitive and blocking ELISAs are very similar. Assays in which the serum being tested and a competing antiserum (or antibody preparation) are reacted simultaneously with the antigen are called competitive ELISAs. Assays in which the serum being tested and the competing antiserum (or antibody preparation) are reacted sequentially with the antigen are called blocking ELISAs.

Antigen capture ELISAs are powerful tools for the demonstration of antigens in samples. They are sensitive, specific, cheap and results can be obtained in a few hours and can be done with simple equipment in non-specialist laboratories.

The following topics will be covered in this sub-module:

  • Indirect ELISA
  • Variations of the indirect ELISA
  • ELISA antibody response curves
  • Calculation of results
  • Protocol for a typical indirect ELISA
  • Competitive and blocking ELISAs
  • Protocol for a typical competitive ELISA
  • Antigen capture ELISAs

About The Instructor

Dr Jannie Crafford

Dr Jannie Crafford

  • BVSc, MSc
  • Senior lecturer, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria
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This Work, Enzyme-linked immunosorbent assay (ELISA), by Dr RW Worthington is licensed under a Creative Commons Attribution license.