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Title: Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi

Abstract

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 othermore » 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.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Bulb Research Centre, Lisse (NL)
Sponsoring Org.:
Academy of Finland; National Science Foundation (NSF); USDOE
OSTI Identifier:
20000242
DOE Contract Number:  
FG02-93ER20097
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 65; Journal Issue: 9; Other Information: PBD: Sep 1999; Journal ID: ISSN 0099-2240
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; PLANT DISEASES; MONOCLONAL ANTIBODIES; IMMUNOASSAY; LIPOPOLYSACCHARIDES; DIAGNOSIS; ANTIGEN-ANTIBODY REACTIONS

Citation Formats

Doorn, J. van, Ojanen-Reuhs, T., Hollinger, T.C., Reuhs, B.L., Schots, A., Boonekamp, P.M., and Oudega, B. Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi. United States: N. p., 1999. Web.
Doorn, J. van, Ojanen-Reuhs, T., Hollinger, T.C., Reuhs, B.L., Schots, A., Boonekamp, P.M., & Oudega, B. Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi. United States.
Doorn, J. van, Ojanen-Reuhs, T., Hollinger, T.C., Reuhs, B.L., Schots, A., Boonekamp, P.M., and Oudega, B. Wed . "Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi". United States.
@article{osti_20000242,
title = {Development and application of pathovar-specific monoclonal antibodies that recognize the lipopolysaccharide O antigen and the type IV fimbriae of Xanthomonas hyacinthi},
author = {Doorn, J. van and Ojanen-Reuhs, T. and Hollinger, T.C. and Reuhs, B.L. and Schots, A. and Boonekamp, P.M. and Oudega, B.},
abstractNote = {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.},
doi = {},
journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 9,
volume = 65,
place = {United States},
year = {1999},
month = {9}
}