Isolation of monoclonal antibodies reacting with peribacteriod membranes and other components of pea root nodules containing Rhizobium leguminosarum |
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Authors: | D J Bradley E A Wood A P Larkins G Galfre G W Butcher N J Brewin |
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Institution: | (1) John Innes Institute, Colney Lane, NR4 7UH Norwich, UK;(2) Monoclonal Antibody Centre, AFRC Institute of Animal Physiology, Babraham, CB2 4AT Cambridge, UK;(3) Present address: Celltech, 244 Bath Road, SL1 4DY Slough, UK |
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Abstract: | Plant and bacterial antigens contributing to nodule development and symbiosis in pea (Pisum sativum L.) roots were identified after isolation of a set of monoclonal antibody (McAb)-producing hybridoma lines. Rats were immunised with the peribacteriod material released by mild osmotic shock treatment from membrane-enclosed bacteroids of Rhizobium leguminosarum bv. viceae. In order to diversify the range of McAb specificities, this material was either used as immunogen directly (method 1), or after immunodepletion of a set of glycoprotein and lipopolysaccharide antigens (method 2), or after deglycosylation (method 3). After fusion and screening of cloned hybridoma lines, these three immunisation methods gave respectively 4, 2 and 1 classes of McAb with unique antigen specificities. Ultrastructural immunogold localisation studies showed four different antigens to be present on peribacteriod and plasma membranes (identified by MAC 64, 202, 206 or 209); in addition, a glycoprotein of plant origin but present in the infection-thread matrix was identified by MAC 204. Although none of the epitopes recognised by these McAb was nodule-specific, several were found to be more abundant in extracts of nodule tissue than in uninfected roots (MAC 64, 202, 204, 206). Two McAb reacted with new bacterial antigens: MAC 203 identified a bacterial antigen expressed upon infection but not in free-living cultures of Rhizobium, and MAC 115 identified a bacterial polypeptide (55 kdaltons) that was present in both free-living and bacteroid forms. There were also some McAb of broader specificity that react with antigens present in both plant and bacterial cytoplasms.Abbreviations ELISA
enzyme-linked immunosorbent assay
- Ig
inmunoglobulin
- kDa
kilodalton
- LPS
lipopolysaccharide
- McAb
monoclonal antibody
- PBM
peribacteroid membrane
- SDS-PAGE
sodium dodecyl sulfate-polyacryl-amide gel electrophoresis
- TFMS
trifluoromethane sulfonic acid |
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Keywords: | Glycoprotein Infection thread matrix glycoprotein Lipopolysaccharide Monoclonal antibody Pisum (root nodule) Plasma membrane glycoprotein Rhizobium Root nodule |
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