Antibody Screening Using a Biophotonic Array Sensor for Immune System Response Profiling

279 Pages·2014·9.86 MB·English

Antibody Screening Using a
Biophotonic Array Sensor for
Immune System Response Profiling

Submitted by Thomas Read to the University of Exeter
as a thesis for the degree of
Doctor of Philosophy in Biological Sciences
December 2013

This thesis is available for Library use on the understanding that it is copyright
material and that no quotation from the thesis may be published without proper
acknowledgement.

I certify that all material in this thesis which is not my own work has been
identified and that no material has previously been submitted and approved for
the award of a degree by this or any other University.

Signature: …………………………………………………………..

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 details the assessment of an ‘in house’ built label-
free 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.
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Acknowledgments, Sincerely
I would like to thank my supervisor, Professor Andrew Shaw, whose attention to
detail drove me to finally learn to punctuate prose and almost that data is (ARE)
in fact plural. The amount of times he must have felt like I was the macaque I
dread to think. Special thanks must go to Dr Rouslan Olkhov for his continuous
guidance and support throughout my PhD. I honestly would not have managed
this process without his detailed explanations of anything and everything.
Specific acknowledgment must go to him also for writing the Global Fit script in
Matlab and explaining the principles time and time again. Thanks also to DSTL
(Porton Down, UK) and especially Dr Di Williamson for her advice and all
macaque related consumables.
I would also like to thank Anthony Gregory, who has been through the same
process as I from year one of our undergraduate degree, I feel we have kept
each other sane, especially towards the end. Thanks also to Lynsey Penwill
who was always there to take the focus of myself when things were not going to
plan and Dr Bernard Van Vuuren and Dr Nick Walker, who proved this was all
possible; what now seems like a life time ago.
Also, I would finally like to thank my family, for never doubting me, Stephie for
her continued support throughout this process and all Exeter related friends for
taking my mind off science when needed.

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Table of Contents
1 Introduction 1
1.1 Biomarker Panels and Diagnosis .......................................................... 3
1.2 Biosensors ............................................................................................ 6
1.2.1 Enzyme-Linked Immuno-Sorbant Assays ....................................... 7
1.3 Label vs. Label-Free Biosensors ........................................................... 8
1.3.1 Surface Plasmon Resonance ......................................................... 8
1.3.2 Localised Surface Plasmon Resonance ....................................... 10
1.4 Biospecificity: the Antibody – Antigen Interaction ................................ 12
1.4.1 Antibody Structure ........................................................................ 13
1.5 The Immune System Response and the Production of Antibodies ..... 16
1.6 Immunoassays .................................................................................... 18
1.6.1 Vaccines and the Immunological Response ................................. 19
1.7 Aims and Objectives............................................................................ 20
2 Assessment of the Array Reader Particle Plasmon Biosensor Performance
24
2.1 Introduction ......................................................................................... 24
2.1.1 Assay Performance Metrics .......................................................... 25
2.2 Aims and Objectives............................................................................ 28
2.3 Materials .............................................................................................. 28
2.4 In-House, Label-Free Localised Plasmon Sensing Array Technology 29
2.4.1 Array Nanoparticle Synthesis ....................................................... 29
2.4.2 Array Spot Nanoparticle Characterisation ..................................... 31
2.5 The Array Reader Instrument .............................................................. 32
2.5.1 Sensitivity Analysis ....................................................................... 33
2.5.2 Nanoparticle Surface Activation and Bio-Functionalisation ........... 37
2.6 Immuno-Kinetic Assay Analysis .......................................................... 40
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2.6.1 Langmuirian Analysis of the Immuno-Kinetic Assay ..................... 41
2.7 Langmuir Adsorption Isotherm – Determination of the Kinetic
Parameters ................................................................................................... 44
2.7.1 Fitting 1:1 Binding Interaction Model – the Global Fit ................... 45
2.7.2 Error Propagation in the Global Fit Process ................................. 47
2.7.1 Evaluation of the Goodness-of-Fit for the Global Fit Model .......... 50
2.8 Deviations from the 1:1 Binding Interaction Model .............................. 55
2.8.1 Reducing Non-Specific Binding .................................................... 55
2.8.2 Surface Avidity .............................................................................. 58
2.8.3 Mass Transport Effects ................................................................. 59
2.9 Performance of the Array Reader Technology – Conclusions ............. 60
3 Assay Enhancement and Amplification Based on an Antibody Bio-Stack
Technique......................................................................................................... 61
3.1 Introduction ......................................................................................... 61
3.1.1 Limits of Detection for SPR Instruments ....................................... 61
3.1.2 Continuous vs. Particle: Instrument Contrast ................................ 62
3.1.3 Continuous Surface vs Particle: Assay Contrast........................... 63
3.1.4 Bio-Noise Amplification ................................................................. 65
3.1.5 Amplification Techniques in LSPR ................................................ 66
3.2 Aims and Objectives............................................................................ 68
3.3 Materials .............................................................................................. 69
3.4 Experimental Methods ......................................................................... 69
3.4.1 Array Reader Particle Plasmon Surface ....................................... 70
3.4.2 SensiQ Propagating Plasmon Surface ......................................... 72
3.5 Results ................................................................................................ 73
3.5.1 Antibody Amplification Bio-Stacking ............................................. 73
3.5.2 Peanut-Specific Antibody Screening in Peanut Allergy Patient
Serum 78
3.6 Discussion ........................................................................................... 80
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3.6.1 Penetration Depth ......................................................................... 81
3.6.2 Amplification via Antibody Oligomerisation Bio-Stacking .............. 85
3.7 Conclusion .......................................................................................... 92
4 Total IgG Concentration Assay .................................................................. 94
4.1 Introduction ......................................................................................... 94
4.1.1 Current Assays for Measuring Total IgG ....................................... 96
4.1.2 Antibody Fc Region ...................................................................... 98
4.1.3 Bacterial Immunoglobulin Binding Proteins ................................ 100
4.1.4 Chimeric Fusion Protein A/G ...................................................... 104
4.2 Aims and Objectives.......................................................................... 105
4.3 Materials ............................................................................................ 105
4.4 Experimental Methods ....................................................................... 106
4.4.1 Assay Surface Functionalisation ................................................. 106
4.5 Results .............................................................................................. 107
4.5.1 Purified Antibody Binding Characteristics ................................... 107
4.5.2 Human Serum Total IgG ............................................................. 111
4.5.3 Immunoglobulin Isotype Specificity ............................................. 112
4.6 Discussion ......................................................................................... 114
4.6.1 Purified Polyclonal Antibody Binding Kinetics ............................. 114
4.6.3 Immunoglobulin Isotype Specificity Analysis .............................. 125
4.7 Conclusion ........................................................................................ 129
5 Optimisation of Protein Screening Assays and their Application in Clinical
Settings .......................................................................................................... 132
5.1 Introduction ....................................................................................... 132
5.1.1 C-Reactive Protein ...................................................................... 133
5.1.2 Glutaraldehyde Protein Cross-linking ......................................... 136
5.2 Aims and Objectives.......................................................................... 138
5.3 Materials ............................................................................................ 139
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5.4 Experimental Method ........................................................................ 139
5.4.1 Surface Preparation .................................................................... 139
5.4.2 ‘in situ’ Surface Preparation ........................................................ 140
5.5 Results .............................................................................................. 142
5.5.1 CRP-Assay ................................................................................. 143
5.6 Discussion ......................................................................................... 146
5.6.1 Conclusion .................................................................................. 153
6 Evaluating the Immune Response of a Yersinia pestis vaccine in a
Macaque Animal Model .................................................................................. 154
6.1 Introduction ....................................................................................... 154
6.2 Aims and Objectives.......................................................................... 159
6.3 Materials ............................................................................................ 159
6.3.1 Sub-unit Antigens and IgG mAbs................................................ 160
6.3.2 Animals and Immunogenicity Studies ......................................... 160
6.4 Experimental Methods ....................................................................... 161
6.4.1 Surface Preparation .................................................................... 161
6.4.2 aLcrV Monoclonal Antibody Profiling .......................................... 162
6.4.3 Macaque Sera Specific IgG Profiling .......................................... 162
6.4.4 Total IgG Determination .............................................................. 162
6.4.5 Constructing the Standard Curves .............................................. 162
6.5 Results .............................................................................................. 164
6.5.1 aLcrV mAb Profiling .................................................................... 165
6.5.2 Macaque Sera Study .................................................................. 167
6.5.3 IgG Calibration of the Serum Sample Data with Monoclonal IgG:
the Epitope Density Factor ....................................................................... 172
6.5.4 Secondary Detection Analysis of the Serum Samples with aIgG 175
6.6 Discussion ......................................................................................... 176
6.6.1 Monoclonal Antibody Study ........................................................ 177
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