M.F. Clark

Enzyme immunosorbent assays represent a departure from the classical procedures based on immunoprecipitin reactions in that recognition of immunospecific activity is through the action of the associated enzyme label rather than by observing the formation of an insoluble antigen–antibody complex. By using an enzyme marker, usually linked to the virus-specific antibody, the detection of a specific reaction can be augmented several hundredfold relative to the threshold of visibility of an immune precipitate. Essentially, the same principle is utilized also in methods involving radio- or fluorescent labels and in serological electron microscopic techniques in which antibodies are “tagged” with electron-opaque markers. Most applications of enzyme immunosorbent assay for the detection of plant viruses have utilized the double antibody sandwich (DAS) method of enzyme-linked immunosorbent assay (ELISA) (Clark and Adams, 1977) in which specifically immobilized viral antigen is allowed to react directly with enzyme-labeled antibody, the resultant complex being revealed by the addition of enzyme substrate. Protocol N lists the sequence of operations for a typical routine assay of virus in field-collected samples, using this form of ELISA. Procedures in use in plant pathology fall into one of two main categories: direct procedures, in which antigen immobilized on the solid phase is detected with an enzyme-labeled, specific antibody, and indirect procedures, in which the immobilized antigen is the target for unconjugated, specific antibody, which in turn is detected by an enzyme-labeled, anti-immunoglobulin molecule.

Chapter 3