Development of novel antibody-based diagnostics for the early and rapid detection of cardiac markers

The principal objective of this research was the production, characterisation and application of antibodies for the detection of both C-reactive protein (CRP) and Myeloperoxidase (MPO), two promising candidates in the field of cardiac diagnostics. CRP is a highly stable acute phase reactant, produced in the liver in response to interleukin (IL)-6. In January 2003 both the Centres for Disease Control and Prevention (CDC) and the American Heart Association (AHA) recommended CRP as the inflammatory marker of choice for assessment of cardiovascular risk. The other biomarker under investigation was MPO, a mediator enzyme secreted by inflammatory cells such as activated neutrophils and monocytes. High levels of MPO have been reported in atherosclerotic plaques and accumulating evidence indicates that MPO may have a causative role in plaque vulnerability. It has been suggested that these markers may even be complementary in the assessment of patient cardiac risk. As a marker of disease activity and vascular inflammation, CRP detection is associated with longterm risk stratification while MPO, being a marker of plaque instability and neutrophil activation, may be prove to be a key marker for short term stratification. A key feature of this work was to ensure the isolated antibodies had a sufficient level of both functional affinity and specificity for the development of novel diagnostic formats and assay platforms. Both classic polyclonal antibody generation and ‘state-of-the-art’ phage display technology were employed for the generation of CRP- and MPO-specific antibodies and recombinant fragments. The immune systems of mice, rabbits and chickens were investigated in this work, which allowed for the identification of the main advantages and disadvantages associated with each of the host immune systems and their capacity for generating specific and sensitive antibodies. It was observed that the avian immune system was found to be the most effective system for the generation of functional and soluble antibodies. The CRP-specific antibodies were incorporated into three novel biosensor platforms for their final characterisation. In addition, a novel lateral flow immunoassay (LFIA) for the rapid detection of MPO was developed, using a recombinant antibody isolated from a MPOimmune library. Spiked samples of depleted human serum were employed in both the biosensor and LFIA systems, thus validating the detection of the analytes in a realistic sample matrix

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

Dublin City University

Year dc:date.issued

2010

Author dc:creator

• McDonnell, Barry

• Health

Language dc:language

en