Abstract
dc:description.abstractAcute Kidney Injury is a common condition seen in 30% of neonates in the ICU and results in poor healthcare outcomes. The rapid assessment of the glomerular filtration rate (GFR), a metric of kidney function, can guide the dosing of renally-cleared lifesaving antibiotics, thereby preventing organ damage due to drug toxicity. GFR estimation is routinely done by measuring changes in creatinine levels which aren’t rapid enough to inform kidney dysfunction in neonates. Changes in Cystatin C (CysC) serum levels are physiologically time effective, but current testing methods precludes the usefulness of CysC as a fast responder. This thesis explores the design of bio-reagents that could be used in the development of a ‘faster’ point-of-care immunoassay to measure serum CysC. By engineering CysC-specific single chain variable fragment (scFv) antibodies with a cellulose binding tag (CipA) to tether it to paper, and a fluorescent protein (mCherry) to provide a detectable signal, the aim is to develop a paper-based sandwich immunoassay that can be used at bedside for faster results. Using phage-display screening, 14 scFv antibodies specific to CysC, were obtained. They were successfully expressed in an E. coli host system, and they exhibited good solubility, thermal stability, and high binding affinities to CysC. One scFv was engineered to incorporate CipA and mCherry at different locations with respect to the scFv and the fusion effects of tag addition were studied. All configurations retained their native CysC-binding activity, cellulose binding capacity and fluoresced well. The engineered scFvs showed remarkable stability in-solution as well as on cellulose, providing practical value in its utility. Lastly, the 14 scFv clones were analysed based on their epitope specificity, first computationally and then experimentally, to find a pair that binds CysC non-complementarily to facilitate sandwich formation. The inherent analytical metrics of these native and engineered-scFv clones in assessing neonatal CysC serum concentrations were explored, providing insights for further development and use in guiding clinical decisions. The assay developed is self-labelling without the need for chemical crosslinking of labels, can be developed into a sustainable cellulose-based point-of-care sensing platform, and shows the modularity and usefulness of engineered scFv in diagnostic platforms.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Manohar, Madhuri
- Advisors dc:contributor.advisor
-
- Hall, Elizabeth
- Higson, Seamus
Subjects
dc:subject × 4Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.112619
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/374648