The development of monoclonal antibodies to the secA protein of Cape St. Paul wilt disease phytoplasma and their evaluation as a diagnostic tool.
Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK, jennifer.hodgetts@fera.gsi.gov.uk.
Jennifer
J
Hodgetts
Gaynor
G
Johnson
Kate
K
Perkins
Sioban
S
Ostoja-Starzewska
Neil
N
Boonham
Rick
R
Mumford
Matthew
M
Dickinson
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D003001:Cloning, Molecular; D018604:Epitope Mapping; D007114:Immunization; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D045582:Phytoplasma / Q000201:enzymology* / Q000276:immunology; D010935:Plant Diseases / Q000276:immunology* / Q000382:microbiology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins