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Ip Innovative Publication Pvt Ltd Elisa and Related Techniques by R S Chauhan and Ankita Joshi
It is a matter of great pleasure and satisfaction to write this monograph on ELISA and Related Techniques. Enzyme linked immunosorbent assay (ELISA) is a widely applicable and accepted test routinely used now-a-days for diagnosis and confirmation of infectious diseases. It has the same principle and sensitivity as radioimmunoassay (RIA) and replaced the later being less expensive and safe in handling during procedure. In radioimmunoassays, the use of radioisotope labelled antibodies or antigen are hazardous to the health and it requires the costly gamma or beta counter to interpret the results. The basic principle of ELISA is same as RIA but in place of radioisotope labelled antibodies, in ELISA an enzyme is used. This assay is also used for the detection of antigen as well as antibodies. For detection of antibodies, the known antigen is coated on the surface of well of polystyrene plate (96 wells) and then test serum samples, properly diluted, is added. After incubation, the plates are washed and conjugate enzyme labelled species specific antibody is added which binds with the antibodies present in test serum and can be detected by adding substrate which gives a coloured reaction that can be seen by naked eyes or read in spectrophotometer. For detection of antigen, the sandwich assay is preferred. In sandwich assay, antibody is bound to the solid phase (polystyrene plate). This antibody is known as capture antibody. Then the specimen suspected for presence of antigen is added. After appropriate incubation, the plate is washed and second antibody specific to antigen is applied, which is conjugated with enzyme in direct method while in indirect method this antibody is plain and anti-antibody conjugated with enzyme is applied which is known as indicator antibody. In indirect method the secondary antibody should be raised in a different animal species with that of capture antibody. The reaction is detected by applying enzyme substrate, which gives coloured reaction.