In praise of the ELISA

11th December 2014

Posted By Paul Boughton

Illustration of the ELISA process

Michael Childerstone pays tribute to the remarkable role of the ELISA in the life sciences industry

ELISA – the enzyme linked immunosorbent assay – is one of the unsung heroes of the life science laboratory. Radioimmunoassay, the technique for which Rosalind Yalow was awarded the Nobel Prize for medicine in 1977, has largely been replaced by immunoassays of the type that use enzyme or fluorescent labels. Of these, ELISA based on the 96 well microplate is by far the most widespread.

Not so very much has changed since the first description of the technique by Engvall and Perlman in 1971 with their assay for human IgG. The first ELISAs utilised polystyrene test tubes onto which an antibody or antigen was passively adsorbed onto the surface.

The sample to be tested was incubated in the tube and after rinsing, some sort of probe that had been chemically conjugated to an enzyme was added enabling detection of the analyte by a conventional enzyme assay with a coloured end point (see Fig. 1).

The test tube was soon replaced by the familiar 96-well microplate, enabling 96 ‘test tubes’ to be manipulated as one unit. Initial problems with the uniformity of binding across the 8 x 12 array have largely been solved by industry and several companies offer products with a variety of binding characteristics.

With some of the high binding surfaces a wider range of detergents can now be used but by and large, Tween 20 is still the most popular, as it successfully treads a fine line by washing effectively whilst leaving the sensitised surface intact. Alkaline phosphatase and horseradish peroxidase were the two enzymes used to prepare conjugates from the beginning and remain the most popular today.

The protocol can be varied to enable detection and measurement of both small and large molecules, such as drugs, metabolites, hormones, cytokines, and the antigens of viruses, bacteria and parasites.

The assay is especially suitable for detecting specific antibodies and has a very wide range of uses, not only for the serological diagnosis of disease but also to screen new monoclonal antibodies.

Three pieces of laboratory kit enable the ELISA to be performed efficiently. These are an eight-channel micropipette, a microplate washer and a microplate photometer. The widespread use of ELISA means that these items of equipment are readily available and will be found in many life science laboratories.

As thousands of different ELISAs are described in the literature and hundreds are commercially available in kit form it would be easy to overlook the fact that the demand for new or better assays has not diminished over the past 43 years. 

With some background in the tricks and pitfalls involved in assay development it is quite possible to get the beginnings of an assay up and running within a few days once the antibodies have been produced (or purchased) – clearly much more work is required to complete and validate a new assay. 

The simplicity in the technique also means that very often ELISA is the starting point for testing new reagents and for prototyping the many other kinds of immunoassay in use today.

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Michael Childerstone is director of Kalon Biological, in Guildford, UK. 





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