Indirect ELISA

∞ generated and posted on 2016.03.20 ∞

Serological procedure involving detection of antigen via the binding of enzyme-linked antibody to an antibody that has already been specifically bound to the antigen.

An indirect ELISA, also known as a sandwich ELISA, is indirect in the sense that the enzyme-linked antibody does not bind directly to the antigen, but instead binds to a different antibody that first has been bound directly to the antigen. That is, in an indirect ELISA the linkage between antibody and enzyme involves two rather than just one intervening antibody. Recall that ELISA stands for Enzyme-Linked ImmunoSorbent Assay.

A utility of indirect ELISAs is that one need not chemically enzyme link the antibodies supplying specificity to the assay, but instead can have on hand or, rather, can easily purchase enzyme-linked antibodies that have a broader utility. That is, the specificity of these other antibodies can be for a breadth of antibodies, each of which supplies a different specificity, and therefore enzyme-linked antibody can be useful under different circumstances.

The antibody that supplies the specificity and the antibody that supplies the enzyme are not only two different reagents but they supply two different specificities. It is like having a master key that opens many locks (the enzyme-linked antibody) rather than employing a different key to open each individual door in a building.

Indirect ELISAs may be used particularly for small-scale ELISAs that will be employed relatively few times. By contrast, one employs direct ELISAs for larger scale procedures which justify enzyme linking the specificity-supplying antibody en masse.

Indirect ELISAs are possible because the two different antibodies involved are sourced from different species. The first antibody, that directly binds to the antigen, is from one species, such as a mouse. The second antibody is formed via the immunization of a different species with the first species' antibody, creating, for example, rabbit anti-mouse antibody. The latter antibody is then chemically linked to the enzyme and its specificity is for the antibody that has specificity for the antigen, that is, rather than directly for the antigen which is being assayed by the ELISA.