Radioimmunoassay, enzyme and non-enzyme-based immunoassays

Abstract

The ability to quantify the amount of a specific protein in a complex sample has been a valuable addition to laboratory science, allowing the development of diagnostic tests, allergen detection in the food industry,and screening for immunity. This is particularly important in anaesthesia, intensive care, and pain research for the quantification of mediators (cytokines, peptides, and analytes) involved in inflammation, pain, and other pathways. Immunoassays use the high specificity of antibodies, along with their enormous diversity, to target specific molecules of interest and analyse their concentration in a sample. The first immunoassay developed was described by Yalow and Berson in 1959. They used radiolabelled insulin to assess the concentration of insulin in human plasma, and thus developed the first radioimmunoassay (RIA). In 1971, Engvail and Perlman described a technique whereby antigens were immobilized on a microplate well, incubated with antiserum, and then the concentration of antibody in the antiserum was quantified using an enzyme-linked anti-immunoglobulin antibody. This method is the enzyme-linked immunosorbent assay (ELISA). Enzyme immunoassays (EIAs) are very similar to ELISAs, and as such, the terms are often used interchangeably. The EIA was developed by Van Weemen and Schuurs (independently of Engvail and Perlman) for the quantification of antigen rather than antibody. For the purpose of this article, EIA and ELISA should be considered interchangeable. The majority of RIA assay formats recommend sample cleaning and concentration (particularly when analyte concentration and assay sensitivity is low), although a large number of ELISA assays can cope with direct use of unprocessed plasma. The cleaning and concentration process usually involves ion exchange chromatography followed by some form of freeze drying/lyophilization. We would recommend users to determine if sample cleaning is required for their analyte. Often, there are differences in measured analyte concentration when comparing RIA and ELISA. This can result from specificity of the antibody (e.g. the cardiovascular peptide urotensin II) 6 or the fluid in which the analyte is suspended interfering with only one type of assay (e.g. the opioid-related peptide Nociceptin/Orphanin FQ). – 11 Discordance has also been demonstrated between RIAs and EIAs measuring cortisol and carcinoembryonic antigen. 13 The selection of assay format is therefore critical and the remainder of this article covers the main formats currently available.