RESULTS:

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.

The use of enzyme-linked antibodies for the detection of two morphologically different plant viruses is described. The technique is extremely sensitive enabling assay of the viruses at concentrations as low as 10 to 100 ng/ml both in purified preparations and in crude plant extracts.

Preparations of alfalfa mosaic virus made by precipitation with polyethylene glycol were as infective and contained the same relative proportions of components as virus preparations made by ultracentrifugation. A column chromatographic procedure using a continuously decreasing concentration gradient of polyethylene glycol was employed to fractionate the nucleoprotein components of the virus. By this procedure a partial sorting of the virus components into three groups according to particle length was achieved. Particles in the three groups are thought to correspond to top component a top component b and a mixture of middle and bottom components.