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Title: Identification of the causative agent of Kawasaki disease through isolation and characterization of antigens within immune complexes
Author: Menikou, Stephanie
ISNI:       0000 0004 7657 3518
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Kawasaki disease (KD) is an inflammatory disease in children that is the most common cause of paediatric acquired heart disease in developed countries. The cause of KD is unknown but epidemiological data implicate an infectious agent. The presence of immune complexes (ICs) in the serum of children with KD suggests that immunological processes are important in KD pathogenesis. This study investigated the hypothesis that the causative agent of KD could be identified by IC recovery and sequencing of antigens within the ICs in sera from children with KD. After reviewing the methods available for IC recovery, the first approach used was the well-established method for IC detection and recovery, Polyethylene glycol (PEG) precipitation. Although PEG precipitation is a simple method, and has the advantage of not denaturing proteins, newer protein recovery and separation approaches were considered in order to recover ICs from larger volumes of serum. A novel approach to recover and identify ICs was developed using size exclusion chromatography (SEC) and affinity chromatography on immunoglobulin binding columns (HiTrap Protein G). The purification process was monitored by 1D SDS-PAGE, protein staining, Western blotting and, finally, liquid chromatography tandem mass spectrometry (LC MS/MS) was used to identify the recovered antigens. This approach was first applied to serum with artificially created vaccine antigen (influenza)-antibody complexes, which led to recovery and identification of influenza peptides within the recovered IC's. Subsequently, a pilot study was undertaken using a pool of KD serum samples (n=19) and healthy controls (n=4). LC MS/MS results suggested that it was possible to capture immunoglobulins and possibly any antigen(s) in complex with the IgG. In a validation study of 10 KD patients, single peptides from proteins of candidate bacteria were identified, e.g. from Rhizobium species, but are of uncertain significance at present. In conclusion, it is possible to recover and analyse ICs using this new pipeline although further substantive study using new samples and optimised methodology is now required.
Supervisor: Levin, Michael ; Langford, Paul ; Hamilton, Shea Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral