title: Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile.
abstract: Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291-a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.
authors.affiliation: School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Ali Nazari
authors.initials: AN
authors.lastname: Shirvan
authors.affiliation: School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom. Electronic address: rob.aitken@glasgow.ac.uk.
authors.forename: Robert
authors.initials: R
authors.lastname: Aitken
mesh_terms: D000907:Antibodies, Bacterial / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D016360:Clostridioides difficile / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003015:Clostridium Infections / Q000175:diagnosis / Q000382:microbiology*; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D021521:Ribotyping
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins
keywords: Clostridium difficile; Phage display; Recombinant antibody