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Applied and Environmental Microbiology, September 1999, p. 4171-4180, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of Xanthomonas hyacinthi

J. van Doorn,1,* T. Ojanen-Reuhs,2 T. C. Hollinger,1 B. L. Reuhs,3 A. Schots,4 P. M. Boonekamp,1 and B. Oudega5

Department of Plant Quality, Bulb Research Centre, 2160 AB Lisse, The Netherlands1; Division of General Microbiology, Department of Biosciences, University of Helsinki, SF-00014 University of Helsinki, Finland2; Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 306023; Department of Nematology, Landbouw Universiteit Wageningen, 6700 ES Wageningen, The Netherlands4; and Molecular Microbiology, IMBW/BCA Faculty of Biology, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands5

Received 11 February 1999/Accepted 1 July 1999

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.

* Corresponding author. Mailing address: Department of Plant Quality, Bulb Research Centre, P. O. Box 85, 2160 AB Lisse, The Netherlands. Phone: 31(0)252462121. Fax: 31(0)252417762. E-mail: joop.van.doorn{at}lbo.agro.nl.

Applied and Environmental Microbiology, September 1999, p. 4171-4180, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • van Doorn, J., Hollinger, T. C., Oudega, B. (2001). Analysis of the Type IV Fimbrial-Subunit Gene fimA of Xanthomonas hyacinthi: Application in PCR-Mediated Detection of Yellow Disease in Hyacinths. Appl. Environ. Microbiol. 67: 598-607 [Abstract] [Full Text]  


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