The development of immunoassays for the detection of bovine brucellosis and aflatoxin B1

The research discussed in this thesis focuses on the development and characterisation of immunoassays for the detection of aflatoxin Bi (AFBi), a toxic fungal metabolite, and for the diagnosis of bovine brucellosis, a highly contagious disease of cattle caused by B ru c e lla abortus. An AFBi-specific single chain fragment variable (scFv) was isolated from a preimmunised phage display library and the gene encoding it sub-cloned into a range of different vectors for soluble expression o f monomeric, dimeric and bifunctional scFvs. The genetically-derived scFvs were then applied to the development of competitive ELIS As and BIAcore-based inhibition assays for the detection o f AFBi. A lateral flow immunoassay (LFIA) was developed for the detection of AFBi using an AFBi-specific monoclonal antibody. Each immunoassay format described was suitable for the detection of AFBi, with high levels of sensitivity, specificity and reproducibility achieved. Several immunoassay formats for the diagnosis of bovine brucellosis, in serum samples, were investigated. Four antigens were selected as diagnostic markers for brucellosis and included whole B. abortus cells, a crude cytoplasmic lysate, an 18kDa cytoplasmic protein ( p i8) and a 26kDa periplasmic protein (bp26). Recombinant forms of the p i 8 and bp26 proteins were cloned and expressed using a high-level expression vector in E.coli. Two polyclonal antibodies, directed against whole B.abortus cells and the crude cytoplasmic lysate, were developed and a naive phage display library was used to isolate scFvs directed against the recombinant bp26. Feasibility studies were carried out on the indirect ELISAs incorporating the four antigens and on the sandwich ELISAs with the polyclonal antibodies. The indirect and sandwich ELISAs, for the diagnosis of bovine brucellosis, were then validated using a panel of Brucella - positive and negative serum samples

Fv antibodies to aflatoxin B1 derived from a pre-immunized antibody phage display library system

The production and characterization of recombinant antibodies to aflatoxin B[SUB1] (AFB[SUB1]), a potent mycotoxin and carcinogen is described. The antibody fragments produced were then applied for use in a surface plasmon resonance-based biosensor (BIAcore), which measures biomolecular interactions in 'real-time'. Single chain Fv (scFv) antibodies were generated to aflatoxin B1 from an established phage display system, which incorporated a range of different plasmids for efficient scFv expression. The scFv's were used in the development of a competitive ELISA, and also for the development of surface plasmon resonance (SPR)-based inhibition immunoassays. They were found to be suitable for the detection of AFB[SUB1], in this format, with the assays being sensitive and reproducible

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment