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Abstract

PÁNKOVÁ I., KOKOŠKOVÁ B. (2002): Sensitivity and specificity of monoclonal antibody Mn-Cs1 for detection and determination of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Plant Protect. Sci., 38: 117-124.Monoclonal antibody Mn-Cs1 with a high level of sensitivity and specificity for detection and determination of Clavibacter michiganensis subsp. sepedonicus was prepared. Strain C. m. subsp. sepedonicus NCPPB 3467 (as whole cell antigen and extracellular polysaccharides) was used for immunisation of four mice Balb/c. After cloning and verifying, two stable hybridoma clones were gained. One monoclonal antibody, designated Mn-Cs1, was used in all tests. It reacted intensely with extracellular polysaccharides from homologous antigen (> 0.5 mg/ml), weakly with proteins from cell walls (> 200 µg/ml) and with whole homologous antigen (concentration 10 4 -10 3 cfu/ml) in DAS-ELISA. Monoclonal antibody Mn-Cs1 showed a high level of specificity. It reacted neither with bacterial strains of closely related subspecies of Clavibacter michiganensis (C. m. subsp. michiganensis and C. m. subsp. insidiosus) nor with the saprophytic bacteria Pseudomonas fluorescens and Pantoea agglomerans. 118 Vol. 38, Plant Protection Science -2002 bodies because they are produced to single determinants (DE BOER et al. 1988). They can replace polyclonal antisera in any one of the serological diagnostic procedures. In contrast to data obtained with monoclonal antibodies, those gained with polyclonal antisera were often conflicting and un-convicting due to the occurrence of false positive cross-reactions. The use of monoclonal antibodies, in comparison with polyclonal antibodies, is also complicated by the appearence of cross-reactions with various pathogenic and saprophytic soil bacteria found in potato extracts.Research institutes and laboratories of plant health services dealing with the diagnosis of the ring rot pathogen buy antibodies for immunochemical diagnosis of Cms available on the market and/or prepare their own antibodies (DE BOER et al. 1988;WESTRA et al. 1994). The use of more than one antibody for detection of the target bacterium in plant samples excludes even more potential crossreactions.Individual bacterial pathogens could be more or less heterogeneous in their genetic, pathogenic, immunochemical and biochemical characteristics due to e.g. changing climatic conditions and/or changes in the host plants. It is, therefore, possible that an available antiserum would not identify all strains of one bacterial pathogen. That is why testing new antibodies for Cms against a large panel of strains is always significant.In this study, we have focused on the preparation of a monoclonal antibody for detection and determination of Cms, and on the evaluation of its sensitivity and specificity by DAS-ELISA. MATERIAL AND METHODS Bacteria:Bacterial strain Cms NCPPB 3467 (National Collection of Plant Pathogenic Bacteria, York, Great Britain) was used as a source of antigen for preparati...