ELISA

The enzyme-linked immunosorbent assay (ELISA) was independently reported by Engvall & Perlmann and Van Weemen & Schuurs in 1971. 50 51 It can be used to detect the presence and/or concentration of some substance in a mixture of unknown composition. It was designed to replace radioimmunosorbent techniques (RIST), which used radiolabels, and therefore presented some health and environmental concerns. 50 We will here describe the Engvall/Perlmann method as a way to describe the method as a whole.

General scheme for detection of a specific antigen in a solution using the Enzyme Linked Immunosorbent Assay.
Figure 1: General scheme for detection of a specific antigen in a solution using the Enzyme Linked Immunosorbent Assay. Original figure.

Figure 1 depicts a general scheme for detecting an antigen (red) in a solution using ELISA. First, an antigen is directly affixed to a surface.  In this case anti-rabbit IgG-serum from sheep was affixed to cellulose, and it was probed with an enzyme-linked antibody (light green linked to blue in figure 1). The Engvall and Perlmann scheme had only one antigen in solution, but we depict a second in brown that was not probed, to illustrate the selectivity of the assay. The surface-affixed antigen is called the immunosorbent. In the specific example used by Engvall and Perlmann, the antibody was rabbit immunoglobulin G (IgG), and the enzyme to which it was linked was alkaline phosphatase (ALP).  The enzyme-linked antibody is incubated with the surface-bound antigen, and then the surface is washed. This removes any antibody not bound to surface antigen (none shown in figure 1). Binding of antibody to antigen can then be assessed by reaction with a chromogenic substrate. Engvall and Perlmann used para-nitrophenylphosphate as their substrate (yellow and red in the last step of figure 1), monitoring its hydrolysis by an increase in absorbance at 400nm.50 Some excellent supplementary videos on the topic can be found at: http://www.piercenet.com/method/overview-elisa

 

A wrinkle in time back to: The Answer

Leave a Reply

Your email address will not be published. Required fields are marked *