A simple indirect ELISA using F(ab')2 fragments of immunoglobulin

Abstract

An indirect ELISA is described in which (i) virus is trapped by F(ab')2 fragments of specific IgG immobilized on a solid phase support, (ii) trapped virus is detected by intact IgG (from the same or a different antiserum) and (iii) positive reactions are identified using an enzyme conjugate specific for the Fc portion of IgG. Pepsin digestion of the Fc portion of the trapping antibody permits the use of a general purpose enzyme conjugate to discriminate between trapping and detecting antibody. Consequently, the assay requires only a single virus-specific antiserum which is often all that is available to the plant virologist. The assay was at least as sensitive for detecting small amounts of antigen as the standard double-antibody sandwich procedure and, for some viruses, two- to fourfold more sensitive. The improvement in performance resulted largely from lower and more consistent background reactions. Both assays were equally effective in revealing heterologous reactions when optimized for detecting homologous antigen. However, increased cross-reactions were obtained in the F(ab')2 procedure by the use of higher concentrations of detecting antibody. The assay is considered particularly suited for comparing antisera from different sources or of different bleeds from the same source, and for investigations involving so few tests that the effort or expense of preparing individual virus-specific conjugates is not justified.