Recombinant antibodies against the K99 colonisation factor of E. coli

Golchin, Mehdi (2004) Recombinant antibodies against the K99 colonisation factor of E. coli. PhD thesis, University of Glasgow.

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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.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QR Microbiology > QR180 Immunology
S Agriculture > SF Animal culture > SF600 Veterinary Medicine
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Supervisor's Name: Aitken, Dr. Robert
Date of Award: 2004
Depositing User: Angi Shields
Unique ID: glathesis:2004-4074
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 05 Mar 2013 15:26
Last Modified: 05 Mar 2013 15:26

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