ISHS Acta Horticulturae 193: XIII International Symposium on Fruit Tree Virus Diseases
PROTEIN A - ALKALINE PHOSPHATE IN ELISA DETECTION OF CHERRY LEAF ROLL AND PRUNE DWARF VIRUSES IN CHERRY SEEDS |
| Authors: | J.I. Cooper, M.L. Edwards, J. Bradley |
| Abstract:
The direct double antibody sandwich form of enzyme-linked immunosorbent assay (DAS-ELISA; Clark and Adams, 1977) is highly strain specific and requires the preparation of a different enzyme conjugated Ig for each virus to be tested. To remedy these deficiencies indirect forms of ELISA have been developed, but to eliminate unwanted reactions between general purpose conjugates and virus-specific antibodies which, in DAS-ELISA bind virus to polystyrene or polyvinyl pyrollidone microtitre plates, a variety of strategies have been used (Van Regenmortal and Buchard, 1980; Lommel et al., 1982; Torrence 1980; Barbara and Clark, 1982). We developed a new procedure (PAS-ELISA) in which anti-virus Ig was induced to a specific orientation by a precoating layer of Staphylococcus aureus Protein A which bound IgG to polystyrene or nitrocellulose via the Fc region of the molecule leaving the F (ab1)2 portion of the IgG available to trap viral antigens applied subsequently. Alkaline phosphatase (2.5 mg Type VII S from Sigma) was conjugated to protein A (0.5 mg quoted binding capacity 12.5 mg human IgG per mg from Sigma) using the two step glutaraldehyde method described by Engvall (1978) to give 3 ml of conjugate that was used at an optimal dilution of 1/1000. With the precoating Protein A at 1 μg/ml and a polyclonal rabbit serum prepared against a cherry leaf roll virus (CLRV) isolate from Betula pendula, CLRV isolates from Prunus avium, Juglans regia, Sambucus racemosa and Rheum raponticum in Chenopodium quinoa sap were readily detected in PAS-ELISA. By contrast, only the cherry isolate was detected when serum against birch CLRV at equivalent Ig concentration was used in DAS-ELISA. In PAS-ELISA purified CLRV was detected at 12–24 ng ml whereas DAS-ELISA routinely detected half this amount of viral antigen (Massalski and Cooper, 1984). When infected Nicotiana megalosiphon, Cucumis sativus or C.quinoa were used as sources of antigens, PAS-ELISA was as sensitive as DAS-ELISA in detecting raspberry ringspot, arabis mosaic, prune dwarf, prunus necrotic ringspot or poplar mosaic viruses. As judged by PAS-ELISA, the relative abundance of antigens shared between CLRV were consistent with properties reflected in immunodiffusion serology and varied in step with DNA hybridization analyses made using DNA complementary in sequence to the RNA 1+2 of birch or rhubarb isolates (Massalski, 1984). To evaluate the usefulness of PAS-ELISA for indexing, leaves or seeds of Prunus avium were tested for prune dwarf virus. For leaves, it was necessary to use polyvinyl pyrollidone to diminish A405 nm values in virus free controls from 0.4 to <0.1, but there was no similar need when testing seeds. | |
Download Adobe Acrobat Reader (free software to read PDF files) | |
