ISHS Acta Horticulturae 235: XIV International Symposium on Fruit Tree Virus Diseases
GOLDLABELLED-IMMUNOSORBENT-ELECTRONMICROSCOPY WITH PLUM POX VIRUS SPECIFIC MONOCLONAL ANTIBODIES |
| Authors: | G. Himmler, U. Brix, M. Laimer, D. Mattanovich, H.W.D. Katinger |
| Abstract:
Plum Pox Virus is the causing agent of the sharka disease of stone fruit trees. Although there exist polyclonal ELISA systems (Clark et al., 1976) of high quality for the diagnosis of the disease, we decided to develop monoclonal antibodies (mAb's) against this virus. The monoclonal antibodies should have the advantage of the in vitro production of a constant quality serum and could be used in basic research on the molecular biology of plum pox virus. The mAb's were produced and then they were screeened by ELISA and GISEM (Goldlabelled Immunosorbent Electronmicroscopy) (Himmler et al., 1987 and 1988). The fact that we got mAb's reacting either with coat protein subunits or with virions or with both, respectively, gave us the chance to check the ELISA routine test system by using electronmicroscopy grids as solid support. The second antibody in this system was a gold labelled one instead of an enzyme labelled one. The buffers used in all experiments were the standard ELISA buffer, i.e. 0.1 M carbonate buffer pH 9.6 for coating and phosphate buffered saline with 0.1% Triton X-100 for washing. Sample dilution buffer was washing buffer with 1% polyvinylpyrollidone and 1% bovine serum albumin The direct coating of partially purified PPV (Grüntzig, 1980) onto the grids showed a strong degradation of virus by reaction with an anti-cryptotope mAb (Figure 1). The sandwich-type assay (GISEM) was performed with grids that were precoated with sheep-anti PPV polyclonal serum and with extracts of PPV infected Nicotiana clevelandii. It can be seen from Figures 2–4 that there can be a large amount of degraded (or not yet assembled) virus particles. In this case one will get a strong reaction in ELISA with polyclonal sera or with monoclonal anti-cryptotopes and some anti-metatopes. The corresponding ISEM, if performed without goldlabel (Kerlan et al., 1981) would show little or no virus particles. | |
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