Enzyme Linked Immunosorbent Assay (ELISA)

Abstract

There have been very few developments that markedly affect the need to greatly revise the text from the last version of this book. This is testament to the fact that heterogeneous enzyme linked immunosorbent assays (ELISA) provide ideal systems for dealing with a wide range of studies in many biological areas. The main reason for this success is test flexibility, whereby reactants can be used in different combinations, either attached passively to a solid phase support or in the liquid phase. The exploitation of the ELISA has been increased through continued development of specifically produced reagents for example, monoclonal and polyclonal antibodies and peptide antigens coupled with the improvement and expansion of commercial products such as enzyme linked conjugates; substrates and chromogens; plastics technology and design of microwell plates; instrumentation advances and robotics. However, the principles of the ELISA remain the same. A brief scan of the literature involving ELISA can be used to illustrate the continued success of ELISA. The number of publications with ELISA mentioned in all science areas from 1976 to 2004 is shown in Table 37.1. A fairly constant increase in the number of papers using ELISA methods is indicated. A breakdown of publications according to science areas in five yearly periods from 1980 in Table 37.2 illustrates the versatility in use of the ELISA, as well as highlighting the major areas of use in Medicine and Dentistry; Immunology and Microbiology Molecular biology and Genetics and Biotechnology. It is interesting to note that the earliest exploitation of ELISA was in Immunology and Microbiology and Molecular Biology and Biotechnology, probably reflecting the greatest research areas. Medicine and Dentistry (associated by the search engine) shows the greatest rate of increase in use (probably in the Medical sphere only) from the 1990s. The search results indicate the continued expansion of ELISA in science and there is no reason to believe that this will change even in the face of modern technologies exploiting molecular methods. The analytical and systematic characteristics of the ELISA are ideally suited to diagnosis at the screening level; for surveillance where larger scale sample handling is required and for research. Many of the accepted standard assays in many scientific fields are ELISA based and have replaced other "gold standard" assays. In conjunction with the rapidly evolving use of molecular methods centering on the polymerase chain reaction (PCR) technologies there is the need to use serological confirmatory methods in a dual approach to directly identify and characterise disease agents and to assess disease prevalence through the measurement of specific antibodies or other chemical factors as a result of infection. The use of ELISA methods in testing the environment, and animal, or plant products as safe for human and animal consumption is also a rapidly evolving area for ELISA. ELISA therefore, has been used in all fields of pure and applied aspects of biology, in particular it forms the backbone of diagnostic techniques. The systems used to perform ELISAs make use of antibodies. These are proteins produced in animals in response to antigenic stimuli. Antibodies are specific chemicals that bind to the antigens used for their production thus they can be used to detect the particular antigens if binding can be demonstrated. Conversely, specific antibodies can be measured by the use of defined antigens, and this forms the basis of many assays in diagnostic biology. This chapter describes methods involved in ELISAs where one of the reagents, usually an antibody, is linked to an enzyme and where one reagent is attached to a solid phase. The systems allow the examination of reactions through the simple addition and incubation of reagents. Bound and free reactants are separated by a simple washing procedure. The end product in an ELISA is the development of color that can be quantified using a spectrophotometer. These kinds of ELISA are called heterogeneous assays and should be distinguished from homogeneous assays where all reagents are added simultaneously. The latter assays are most suitable for detecting small molecules such as digoxin or gentamicin. The development of ELISA stemmed from investigations of enzymelabeled antibodies (1-3), for use in identifying antigens in tissue. The methods of conjugation were exploited to measure serum components in the first true ELISAs (4-6). By far the most exploited ELISAs use plastic microtitre plates in an 8 × 12 well format as the solid phase (7). Such systems benefit from a large selection of specialized commercially available equipment including multichannel pipets for the easy simultaneous dispensing of reagents and multichannel spectrophotometers for rapid data capture. There are many books, manuals and reviews of ELISA and associated subjects that should be examined for more detailed practical details (8-21). The key advantages of ELISA over other assays are summarized in Table 37.3. © 2008 Humana Press.