Generation and Application of Antibodies for Detection of Prostate Cancer Biomarker

218 Pages·2012·21.34 MB·English

Generation and Application of
Antibodies for Detection of
Prostate Cancer Biomarker

Julia Zapatero Rodríguez M.Sc.

Thesis Submitted for the Award of Ph.D.

Based on research carried out in The Applied Biochemistry
Group, School of Biotechnology, Dublin City University,
Dublin 9, Ireland.

Supervisor: Prof. Richard O’Kennedy

January 2017
DECLARATION
I hereby certify that this material, which I now submit for assessment on the
programme of study leading to the award of Ph.D. is entirely my own work, that I
have exercised reasonable care to ensure that the work is original, and does not to
the best of my knowledge breach any law of copyright, and has not been taken from
the work of others save and to the extent that such work has been cited and
acknowledged within the text of my work.

Signed:
(Candidate) ID No.:
Date:

ii
“One never notices what has been done; one can only see what
remains to be done”.
— Marie Curie
iii
Acknowledgments
Firstly, I would like to thank my supervisor, Richard O’Kennedy, for his giving me the
opportunity to work with his group and for his guidance throughout, as well as for
his enthusiasm and expertise in the field.
Thanks to all the members of the Applied Biochemistry Group, for their scientific and
technical advice and, more importantly, for all the good moments we spent
together, in and outside DCU. I would like to especially acknowledge Dr. Shikha
Sharma for opening my mind to new research areas and for being a valuable friend.
Shikha, you cannot imagine how much I have missed our coffee breaks, lunches
together and conference trips since you left. I will always consider you my travel
companion (we made it to seven countries in two years–not too bad!) and I know we
will see each other again. Also, I would like to make a special mention of Jessie and
Rory, for keeping me entertained during the long hours in the lab and becoming
friends beyond work, and of Sarah, for staying in contact and working with me even
from the UK.
To all my PROSENSE fellows, who made our meetings so enjoyable and informative, I
cannot imagine a better group than this. In you I have met such wonderful friends
and colleagues. Pedro, you made PROSENSE possible and to you I owe the great
experience of being part of it. Susana, thanks for being such a great host in
Pontypool, showing us around and opening your home to us. Pawan, Kate, Stefan
and Ioulia, it was a pleasure to work with you and share some laughs along the way.
Thank you to the party animals (you know who you are) that were never too tired to
go out after the workshops. I will always keep good memories of our meetings and
hope that someday our paths cross again.
—
También me gustaría agradecer a mis padres por su apoyo y por animarme siempre
a dar lo mejor de mí misma. Por recibirme en España siempre con los brazos abiertos
y por nuestras habituales conversaciones por Skype. No hay palabras para expresar
iv
todo lo que habéis hecho por mí. Espero que estéis orgullosos de tener otra ‘doctora’
en la familia, aunque no sea médico.
A mi querido hermano Miguel, mis tías Julia e Irene, siempre tentándome a ir a la
playa, mis tíos Luis y Javier, y mi tía Lola. Me siento agradecida de tener una familia
maravillosa que se preocupa por mi. A Rebeca, mi amiga canaria en Dublín, gracias
por esas tardes en el NuBar, las fiestas de Halloween y nuestros viajes a Cork.
Gracias a mis amigas de siempre, por demostrarme que, a pesar de la distancia,
siempre puedo contar con ellas. Teresa, Virginia, Elena y Lucía, nuestros reencuentros
siempre fueron el punto fuerte de las Navidades. Marta, Paloma, Esther y Raquel,
siempre me hicisteis sentir en casa cada vez que volvía a Valladolid. Planear ese viaje
a Islandia con Teresa me dio el empujoncito que necesitaba para acabar la tesis y fue
la mejor manera de celebrar mi ‘thesis submission’.
—
Finally, I would like to dedicate this work to my other half, Richard, for being there
day after day during the good and the hard moments. Thank you for making me
laugh every day and for believing in me. You made me happy throughout this time
and kept me optimistic during the writing process. I don’t know how this would have
been possible without you.

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Table of contents
Declaration................................................................................................ ii
Acknowledgements.................................................................................. iv
Table of Contents.....................................................................................vii
List of Abbreviations.................................................................................xi
Units........................................................................................................xiv
List of Figures ......................................................................................... xix
List of Tables..........................................................................................xviii
List of Publications ..................................................................................xix
Abstract................................................................................................... xii

Chapter 1: Introduction........................................................................1

1.1 PROSENSE - THE MARIE CURIE INITIAL TRAINING NETWORK .......... 2
1.2 THESIS SUMMARY ......................................................................... 3
1.3 INTRODUCTION TO PROSTATE CANCER .......................................... 3
1.4 BIOMARKERS FOR PROSTATE CANCER DIAGNOSIS: LIMITATIONS OF
SERUM PSA AND THE SEARCH FOR NEW BIOMARKERS ........................ 6
1.4.1 Candidate protein biomarkers for prostate cancer ..................................................... 7
1.4.2 Next generation diagnostic tools for Prostate Cancer ................................................. 9
1.5 ANTIBODIES AND RECOMBINANT ANTIBODY TECHNOLOGY ........ 14
1.5.1 Introduction to the immune system .......................................................................... 14
1.5.2 Antibody overview ..................................................................................................... 15
1.5.3 Generation of avian recombinant antibodies by phage display ................................ 16
1.5.3.1 Recombinant antibody fragments ...................................................................... 16
1.5.3.2 Phage display ...................................................................................................... 18
1.5.3.3 Panning of phage display libraries ...................................................................... 19
1.5.4 Clinical applications of recombinant antibody fragments ......................................... 20
1.6 BIOSENSORS IN CANCER DIAGNOSIS ............................................ 21
1.6.1 Overview to biosensors ............................................................................................. 21
1.6.2 Biosensors for cancer detection ................................................................................ 24
1.6.2.1 Amperometric biosensors ................................................................................... 25
1.6.2.2 Impedimetric biosensors ..................................................................................... 26
1.7 MICROFLUIDICS PLATFORMS FOR BIOANALYSIS ........................... 27
1.7.1 Microfluidic-integrated biosensors for diagnostics ................................................... 29
1.7.2 Microfluidics for multiplexing .................................................................................... 31
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1.8 AIMS ............................................................................................ 32

Chapter 2: Materials and Methods.....................................................34

2.1 MATERIALS AND EQUIPMENT ...................................................... 35
2.1.1 Equipment .................................................................................................................. 35
2.1.2 Reagents .................................................................................................................... 36
2.1.3 Bacterial strains ......................................................................................................... 37
2.1.4 Antibodies .................................................................................................................. 37
2.1.5 Antigens ..................................................................................................................... 38
2.1.6 Commercial kits ......................................................................................................... 38
2.1.7 Culture Media formulations ....................................................................................... 38
2.1.8 Buffer formulations .................................................................................................... 39
2.1.8.1 General buffers ................................................................................................... 39
2.1.8.2 Buffers for sodium dodecyl sulfate-polyacrylamide gel electrophoresis ............. 39
2.1.8.3 Buffers for Immobilized metal ion affinity chromatography (IMAC)
purification ..................................................................................................................... 40
2.1.8.4 Buffers for magnetic beads bioconjugation ........................................................ 40
2.1.8.5 Other solution formulations ............................................................................... 41
2.2 METHODS .................................................................................... 41
2.2.1 General molecular biological techniques ................................................................... 41
2.2.1.1 Agarose gel electrophoresis ................................................................................ 41
2.2.1.2 Extraction of DNA from agarose gels using NucleoSpin® Gel and PCR
Clean-Up (MN) ................................................................................................................ 41
2.2.1.3 Ethanol precipitation of DNA .............................................................................. 42
2.2.1.4 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) ........ 42
2.2.1.5 Western Blotting ................................................................................................. 43
2.2.2 Animal immunisation for recombinant antibody production .................................... 44
2.2.2.1 Chicken immunization with the ERBB2 extracellular domain ............................. 44
2.2.2.2 Serum antibody titre determination by indirect ELISA ........................................ 44
2.2.3 Generation of an immune chicken scFv library .......................................................... 45
2.2.3.1 Extraction and isolation of total RNA from immunised chicken spleen .............. 45
2.2.3.2 First-strand cDNA synthesis from total RNA ....................................................... 46
2.2.3.3 PCR Primers for the construction of a chicken scFv library ................................. 47
2.2.3.4 Amplification of chicken antibody variable heavy and light chain genes ........... 48
2.2.3.5 Optimisation of MgCl and primer concentration for amplification of DNA ....... 49
2
2.2.3.6 Splice by overlap extension (SOE) PCR using Platinum Taq polymerase ............. 49
2.2.3.7 Plasmid DNA purification and preparation of an E. coli glycerol stock
expressing pComb3XSS ................................................................................................... 51
2.2.3.8 Restriction-digestion of the scFv insert and pComb3XSS vector ......................... 51
2.2.3.9 Ligation of SOE-product into pComb vector ........................................................ 52
2.2.4 Antibody phage display and library panning .............................................................. 53
2.2.4.1 Transformation of E. coli XL1-Blue electrocompetent cells with scFv-
containing plasmid and rescue of scFv-displaying phage ............................................... 53
2.2.4.2 Enrichment of phage library via panning against immobilised HER2 ................. 54
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2.2.4.3 Polyclonal phage ELISA ....................................................................................... 55
2.2.5 Analysis of scFv-expressing single clones ................................................................... 56
2.2.5.1 Soluble expression of antibody fragments .......................................................... 56
2.2.5.2 Soluble Monoclonal scFv ELISA ........................................................................... 57
2.2.5.3 Competitive scFv ELISA for the detection of free HER2 ....................................... 57
2.2.5.4 Sequence analysis of the anti-HER2-specific clones ............................................ 58
2.2.6 Expression, purification and characterisation of anti-HER2 scFv A4 .......................... 59
2.2.6.1 Optimisation of expression conditions for A4 scFv ............................................. 59
2.2.6.2 Immobilised metal affinity chromatography (IMAC) purification ....................... 60
2.2.6.3 Optimisation of assay parameters for HER2 detection in a competitive
format ............................................................................................................................ 61
2.2.6.4 Competitive ELISA for the detection of HER2 using A4 scFv ............................... 61
2.2.6.5 Fluorescence-based competitive ELISA for the detection of HER2 ...................... 62
2.2.7 Isolation and characterisation of polyclonal antibodies to HER2 .............................. 62
2.2.7.1 IgY purification from eggs of an immunised chicken .......................................... 62
2.2.7.2 Competitive ELISA for anti-HER2 polyclonal antibody characterisation ............. 63
2.2.8 Development of a novel centrifugal microfluidic platform for multiplex
biomarker detection ........................................................................................................... 63
2.2.8.1 Centrifugal disc fabrication ................................................................................. 63
2.2.8.2 Centrifugal disc testing ....................................................................................... 64
2.2.8.3 Conventional benchtop sandwich ELISA for the detection of fPSA ..................... 65
2.2.8.4 Conventional competitive ELISA for the detection of HER2 extracellular
domain (ECD) .................................................................................................................. 66
2.2.8.5 Lab-on-a-Disc ELISA protocols ............................................................................ 66
2.2.9 Evaluation of anti-PSA recombinant antibodies by optical and electrochemical
magneto-immunoassay ...................................................................................................... 67
2.2.9.1 Tosyl-modified magnetic particles bioconjugation ............................................. 67
2.2.9.2 Antibody titration for determination of bioconjugation efficiency ..................... 68
2.2.9.3 Colorimetric magneto ELISA ............................................................................... 69
2.2.9.4 Amperometric magneto-immunoassay .............................................................. 70
2.2.10 Immunosensor fabrication and characterization for HER2 detection ..................... 71
2.2.10.1 Fabrication of the immunosensor and electrochemical procedure .................. 71
2.2.10.2 Impedance measurement procedure ................................................................ 73

Chapter 3: Anti-HER2 scFv antibody: Production, Characterisation and
Use for the Development of an Impedimetric Biosensor.....................74

3.1 INTRODUCTION ........................................................................... 75
3.1.1 The HER2 oncogene ................................................................................................... 75
3.1.2 HER family signaling pathways ................................................................................... 76
3.1.3 HER2 overexpression in cancer .................................................................................. 78
3.2 AIMS OF THIS CHAPTER ................................................................ 79

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3.3 RESULTS ....................................................................................... 79
3.3.1 Immunisation of chicken with HER2 ECD and serum antibody titre
determinations ................................................................................................................... 79
3.3.2 Purification of anti-HER2 polyclonal IgY from chicken eggs ....................................... 80
3.3.3 Generation of avian anti-HER2 scFv library ............................................................... 82
3.3.3.1 RNA isolation from immunised chicken spleen lymphocytes and cDNA
synthesis ......................................................................................................................... 82
3.3.3.2 PCR amplification of chicken variable heavy (V ) and light (V ) chains .............. 83
H λ
3.3.3.2.1 Optimisation of MgCl concentration for PCR amplification ....................... 84
2
3.3.3.2.2 Optimisation of primer concentration for V PCR amplification ................. 85
H
3.3.3.2.3 Amplification of V and V with optimised PCR conditions ......................... 85
H λ
3.3.3.3 Splice by Overlap Extension (SOE) PCR to anneal variable heavy and
variable light chain genes ............................................................................................... 86
3.3.3.4 Restriction-digestion and ligation of the SOE PCR product and the
pComb3XSS vector .......................................................................................................... 87
3.3.4 Antibody library screening ........................................................................................ 90
3.3.4.1 Enrichment of phage library via panning ............................................................ 90
3.3.4.2 Soluble expression and monoclonal analysis of single clones by ELISA .............. 91
3.3.4.3 Analysis of putative anti-HER2 clones by competitive ELISA .............................. 93
3.3.4.4 Sequence analysis of selected clones .................................................................. 94
3.3.4.5 Dose-response competitive studies of anti-HER2 clones .................................... 95
3.3.5 Purification and characterisation of anti-HER2 scFv A4 ............................................. 97
3.3.5.1 Purification of A4 scFv by immobilised metal affinity chromatography
(IMAC) ............................................................................................................................. 97
3.3.5.2 Development of a competitive ELISA assay using scFv A4 for the detection of
HER2 ............................................................................................................................... 99
3.3.5.2.1 Optimisation of assay conditions .............................................................. 100
3.3.5.2.2 Evaluation of competitive assay in serum ................................................. 102
3.3.5.3 Fluorescence-based competitive assay ............................................................. 103
3.3.6 Impedimetric biosensor for HER2 detection ............................................................ 104
3.3.6.1 Determination of optimum time duration, temperature and pH for
immune-complexation ................................................................................................. 107
3.3.3.6.2 HER2 measurements using an impedimetric immunosensor ........................ 111
3.4 DISCUSSION ................................................................................ 113

Chapter 4: ‘Lab-on-a-Disc’ for Multiplexed Detection of PSA and HER2
from Whole Blood ........................................................................... 120

4.1 INTRODUCTION .......................................................................... 121
4.1.1 Centrifugal microfluidics .......................................................................................... 122
4.1.2 Dissolvable-film tabs ................................................................................................ 123
4.1.3 Design of the microfluidic ‘flow cell’ ........................................................................ 127
4.2 AIM OF THIS CHAPTER ................................................................ 129
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4.3 RESULTS ...................................................................................... 129
4.3.1 Reduction of assay incubation times ....................................................................... 129
4.3.2 Protein immobilisation on the incubation chamber ................................................ 130
4.3.3 Liquid handling protocol .......................................................................................... 132
4.3.4 Detection of prostate cancer markers: Benchtop vs. Lab-on-a-disc ........................ 133
4.3.3 Interference studies for the parallel running of competitive and sandwich ELISA
on a microfluidic disc ....................................................................................................... 136
4.4. DISCUSSION ............................................................................... 137

Chapter 5: Development of a Single-Chain Antibody-Based Magneto-
Immunosensor for the Detection of Free PSA .................................. 142

5.1 INTRODUCTION .......................................................................... 143
5.1.1 The history of Dynabeads ........................................................................................ 143
5.1.2 Dynabeads for immunoassay ................................................................................... 144
5.2 AIMS OF THIS CHAPTER ............................................................... 145
5.3 RESULTS ...................................................................................... 146
5.3.1 ScAb E1 conjugation to paramagnetic particles .................................................. 146
5.3.2 Optimisation of assay conditions by magneto-ELISA ........................................... 147
5.3.3 Development of an amperometric magneto-immunoassay ............................... 149
5.3.3.1 Cyclic voltammetry ....................................................................................... 150
5.3.3.2 Selection of optimal bioconjugated magnetic particles concentration for
immunosensor .......................................................................................................... 150
5.3.3.3 Amperometric measurements for free PSA detection .................................. 151
5.3.4 Interference studies ............................................................................................ 153
5.4 DISCUSSION ................................................................................ 154

Chapter 6: Overall conclusions .......................................................... 158

6.1 OVERALL CONCLUSIONS .............................................................. 159

Appendices ....................................................................................... 162

Bibliography ....................................................................................166
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