A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens

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SUMMARYFor many species of Oomycetes, the infection of host plants is initiated by motile biflagellate zoospores. In recent years, studies of these zoospores have utilized monoclonal antibodies directed towards a variety of zoospore components. Although only two species of fungi, Phytophthora cinnamomi and Pythium aphanidermatum, have been used for immunization and initial screening, the reactions of these antibodies with over thirty species of fungi in the Peronosporales or Saprolegniales have now been determined. The present paper reviews the methods employed to produce the monoclonal antibodies and to use them to study the biology of the zoospores and the infection process.The lack of a cell wall means that fixation protocols for zoospores for immunization and screening must be chosen carefully so that cell‐surface or intracellular sites will be accessible to the antibodies. The inclusion of glutaraldehyde in the fixative helps keep the zoospore plasma membrane as intact as possible, and screening with cells fixed in the presence of glutaraldehyde selects for antibodies that bind to the surface of the zoospores. Five different patterns of labelling to the zoospore surface have been found. Other antibodies bind with three distinct patterns to the surface of zoospores and/or cysts.The use of formaldehyde alone in the fixative solution allows fragmentation of the plasma membrane and the exposure of intracellular components. In P. cinnamomi attempts to obtain antibodies directed against intracellular antigens were hampered by the presence of an immunologically dominant component that is stored in small vesicles in the zoospore cortex and secreted onto the surface of cysts. This problem was resolved by immunotolerizing mice neonatally before proceeding with immunization 2 months later. Antibodies directed towards a number of novel sites were obtained in this way.Monoclonal antibodies generated by these methods have been used to identify taxonomically specific spore components, to locate surface molecules that might be responsible for the induction of zoospore encystment and to characterize molecules involved in spore adhesion to potential hosts.