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The Development of Monoclonal Antibodies to the secA Protein of Cape St. Paul Wilt Disease Phytoplasma and Their Evaluation as a Diagnostic Tool

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Abstract

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.

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Acknowledgments

This work was performed as part of a Defra Plant Health Division-funded Taxonomic Fellowship (for JH). The authors would like to thank Professor Phil Jones (Rothamsted Research, UK), Dr. Jaroslava Přibylova (Institute of Plant Molecular Biology, Czech Republic), Dr. Joseph Nipah (CSIR, Sekondi, Ghana), Dr. Nigel Harrison (University of Florida, USA), and Professor Assunta Bertaccini (University of Bologna, Italy) for providing samples. Phytoplasmas were held under Defra Plant Health Licence No. PHL 173B/5244.

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Authors and Affiliations

  1. School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK

    Jennifer Hodgetts & Matthew Dickinson

  2. The Food and Environment Research Agency, Sand Hutton, York, YO41 1LZ, UK

    Jennifer Hodgetts, Gaynor Johnson, Kate Perkins, Sioban Ostoja-Starzewska, Neil Boonham & Rick Mumford

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  1. Jennifer Hodgetts
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  2. Gaynor Johnson
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Correspondence to Jennifer Hodgetts.

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Hodgetts, J., Johnson, G., Perkins, K. et al. The Development of Monoclonal Antibodies to the secA Protein of Cape St. Paul Wilt Disease Phytoplasma and Their Evaluation as a Diagnostic Tool. Mol Biotechnol 56, 803–813 (2014). https://doi.org/10.1007/s12033-014-9759-8

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