Characterization of Monoclonal Antibodies against Potato Virus X and Comparison of Serotypes with Resistance Groups

Abstract

SUMMARYThe production and testing of 10 rat monoclonal antibodies specific for strains of potato virus X (PVX) are described. Monoclonal antibodies (MAbs) with a variety of specificities were selected. At one extreme, MAb MAC 58 reacted with all 33 PVX isolates tested ; in contrast, MAb MAC 67 reacted strongly with only two of 33 isolates, one of which was the resistance-breaking PVX-HB against which it was raised. The reactions of the PVX isolates with the MAbs were compared with the classification of PVX strains based on their effects in plants carrying different resistance genes; a preliminary serological classification is suggested. It is possible to distinguish PVX-HB from all other isolates tested using two MAbs (MAC 67 and MAC 59), and these, together with MAC 58, were produced in large quantities in ascitic fluids. Together they provide a valuable set of reagents for use in routine testing.

Characterization of Monoclonal Antibodies to a U.K. Isolate of Barley Yellow Dwarf Virus

“…Antibodies to Swedish isolates 27/77 and 39/78 were from M. Eweida (Eweida & Oxelfelt, 1985). Immunoglobulins (Ig) and alkaline phosphatase enzyme conjugates were prepared by the methods described by Torrance et al (1986).…”

“…Hybrid cell lines were checked for the presence of specific antibody in an ELISA by coating the microtitre plates (Nunc lmmunoplate II) with BYDV preparations followed by tissue culture supernatant fluid and alkaline phosphatase-or horseradish peroxidase-labelled anti-rat IgG using the incubation periods, buffers etc, described by Torrance et al (1986). The direct double antibody sandwich ELISA (DAS ELISA) of virus isolates was done using polyclonal anti-F or -B (1 or 2 gg/ml respectively) to coat the microtitre plates (Nunc …”

“…Ten to 15 days after fusion, fluids from growing cultures were tested for the presence of specific antibody (see below). Selected positive cultures were cloned by limiting dilution as described by Torrance et al (1986).…”

Expression of Potato Virus X Resistance Gene Rx in Potato Leaf Protoplasts

“…Fluorescent antibody staining of infected protoplasts, estimation of virus content by ELISA and infectivity assays were as described by Adams et al (1985). Western blotting was done with a monoclonal antibody to PVX (MAC 72), provided as a tissue culture supernatant by Dr L. Torrance, MAFF, Harpenden, U.K., which detects isolates DX and HB (Torrance et al, 1986). Protoplast samples were prepared essentially as described by Nassuth (1982).…”

A Comparison between the Capsid Proteins and the Products of in vitro Translation of Three Strains of Potato Virus X

“…In order to determine which, if any, of the fragments generated by V8 protease digestion of the three PVX capsid proteins shared a common epitope, the peptides were separated by 15 ~ SDS-PAGE, electroblotted to nitrocellulose (Towbin et al, 1979) and probed with a monoclonal antibody, MAC 72, originally raised against isolate HB and known to react with all three isolates in indirect ELISA (Torrance et al, 1986). The second antibody was an anti-rat antibody conjugated to horseradish peroxidase (Miles Laboratories) and the enzymic reaction was developed as described by de Blas & Cherwinski (1983).…”