K99 was chosen as a model target for this study, to explore the application of recombinant antibody technology to livestock infection. Our aims were to isolate and characterise single-chain Fv antibodies against K99 using phage display. Escherichia coli B41, a clinical isolate that expresses K99 fimbriae, was grown and fimbriae were extracted by heat-shock treatment and then precipitated by ammonium sulphate. The major K99 subunit (Fan C) was purified from crude fimbriae extract by an ion-exchange chromatography method using SP-XL columns. The semi-synthetic Tomlinson I and J libraries (Center of Protein Engineering, Cambridge, UK) were used to select phage antibodies against K99 using immunotubes coated with the major fimbrial subunit. After three rounds of selection, phage were transfected into E. coli HB2151 for the expression of soluble scFvs. Fifteen scFv clones with high activity against K99 fimbriae were identified by ELISA and sequenced. Of these, six scFvs carried sequences that were reasonably diverse. These proteins were purified for further characterization. The recombinant antibodies were shown to react with fimbriae present at the surface of E. coli B41 using immunofluorescence microscopy and immunogold electron microscopy. Some of the purified scFv antibodies were also able to inhibit the agglutination of sheep erythrocytes by E. coli B41 grown at 37°C. To pursue this observation, attempts were made to use the scFvs in an in vitro model of bacterial colonisation in which bacteria were tested for attachment to isolated bovine intestinal villi. Although bacteria could be observed adhering to the brush border, the scFvs appeared unable to prevent this attachment. Further experiments with this in vitro model or a mouse model of ETEC infection, allied with epitope mapping studies should determine if anti-colonisation activity is attributable to binding of scFvs to the receptor-recognition site on the major subunit of the adhesin.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:410017 |
| Date | January 2004 |
| Creators | Golchin, Mehdi |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/4074/ |
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