Antibody Screening Using a Biophotonic Array Sensor for Immune System Response Profile
Read, Thomas
Date: 20 December 2013
Publisher
University of Exeter
Degree Title
PhD in Biological Sciences
Abstract
With a population both increasing in number and age, comes a need for new
diagnostic tools in the healthcare system, capable of diagnosing and monitoring
multiple disorders in a cheap and effective way to provide personalised
healthcare. Multiplex label-free biosensors have the potential to rejuvenate the
current system. This thesis ...
With a population both increasing in number and age, comes a need for new
diagnostic tools in the healthcare system, capable of diagnosing and monitoring
multiple disorders in a cheap and effective way to provide personalised
healthcare. Multiplex label-free biosensors have the potential to rejuvenate the
current system. This thesis details the assessment of an ‘in house’ built labelfree
array screening technology that has potential to be a point-of-care
diagnostic for personalised medicine – the Array Reader.
The performance of the Array Reader platform is considered in detail and
optimised for both antibody and protein screening arrays. A Global Fit protocol
is developed to extract kinetic constants for all protein-protein interactions,
assuming a Langmuir adsorption binding model. Standard operating procedures
are developed to provide optimised dynamic range, sensitivity, reproducibility
and limit of detection of immuno-kinetic assay. A new antibody bio-stack signal
amplification strategy is formed, improving the detection limit 60-fold. As a
consequence, the bio-stack resulted in a novel method for determining the
plasmon field penetration depth, defining the assay sensing volume at the
nanoparticle surface.
Antibody screening arrays were investigated with an IgG quantification assay to
determine total IgG content from serum samples. It relied on the ability of
protein A/G to bind antibodies via the Fc region. Specific antigens were used to
measure the binding properties of the antibody Fab region. By characterising
both regions, we have gained insight into the overall ability of an antibody to
trigger an immune response. Protein screening assay were investigated
targeting C-reactive protein (CRP), a marker of inflammation. The assays
performance characteristics compared favourably with clinically used CRP
assays.
Finally, an antibody screening array was developed to assess the efficacy of a
vaccine against Yersinia pestis in a non-human primate model. The vaccine
screening array is an excellent example of the versatility of the platform and just
one of many possible applications for the future.
Doctoral Theses
Doctoral College
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