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List of protocols xix
Abbreviations xxvii
Preparation of rodent monoclonal antibodies 1 (24)
by In vitro somatic hybridization
Christopher Dean
Philip Shepherd
Introduction and strategy 1 (2)
Choice of host for immunization and myeloma 3 (1)
for cell fusion
Choice of immunogen 4 (1)
Preparation of antigen for immunization 5 (2)
Soluble antigens 5 (1)
Antigens expressed on live cells 5 (1)
Plasmid DNA 6 (1)
Peptides 7 (1)
Route of immunization 7 (2)
Generation of immune spleen cells 7 (1)
Immunization via the Peyer's patches of 7 (2)
rats
Growth of myeloma cell lines 9 (1)
Preparation of cells for fusion 10 (1)
Cell fusion 11 (1)
Screening hybridoma culture supernatants 12 (6)
for specific antibody
Antigen coated multiwell plates 12 (4)
Live or fixed cells 16 (1)
Ligand binding assays 17 (1)
Cloning of hybridomas 18 (1)
Characterization and use of the antibodies 19 (6)
obtained
Isotyping of antibodies 20 (1)
Epitope reactivity 20 (1)
Specific immunoprecipitation 21 (2)
References 23 (2)
Preparation of recombinant antibodies from 25 (33)
immune rodent spleens and the design of their
humanization by CDR grafting
Olivier J. P. Leger
Jose W. Saldanha
Introduction 25 (1)
Preparation of mouse spleen 26 (2)
Isolation of total RNA from spleen 28 (2)
Poly(A+) mRNA isolation 30 (1)
Reverse transcriptase reaction 31 (2)
Primary PCR of antibody genes 33 (4)
Preparation of linker 37 (2)
Assembly of VH and VK gene fragments with 39 (2)
linker DNA
Assembly of single chain Fv antibody 41 (2)
fragments
Reamplification of assembled scFv DNA 43 (1)
Restriction enzyme digestion of assembled 44 (2)
scFv
Purification of pHEN-1 vector by 46 (3)
equilibrium centrifugation in CsCI ethidium
bromide gradients
Restriction digestion of the phage display 49 (1)
vector, pHEN-1
Ligation of pHEN-1 and insert antibody scFv 50 (2)
Preparation of electroporation competent E. 52 (1)
coli TG1 strain cells
Electroporation 53 (2)
Analysis of recombinant clones from the 55 (3)
library
References 57 (1)
Appendix 58 (405)
The design of the humanized antibody 58 (9)
Issues to consider 59 (5)
References 64 (3)
Selection of antibodies from phage libraries 67 (24)
of immunoglobulin genes
Jane K. Osbourn
Introduction 67 (1)
Preparation and storage of phage library 68 (1)
stocks
Phagemid libraries 68 (1)
Phage libraries 69 (1)
Maintenance of bacterial stocks and 69 (2)
titration of phage preparations
Selection of phage libraries on purified, 71 (3)
immobilized antigen
Immobilization of antigen on immunotubes 71 (1)
Elution conditions 72 (1)
Storage and rescue of the phagemid 72 (1)
population after selection
Choice of number of selection rounds 73 (1)
Selection of phage libraries on 74 (2)
biotinylated antigen
Biotinylation of antigen 74 (1)
Selections using biotinylated antigen 75 (1)
Cell surface selections 76 (2)
Adherent cell selections 77 (1)
Cells in suspension 78 (1)
Screening the output of cell surface 78 (1)
selections
Proximity selections 78 (5)
Proximity selection using an existing 80 (1)
antibody
Proximity selection using natural ligands 81 (2)
Step back selections 83 (1)
Screening of selected phage 83 (4)
Basic screening assays 83 (4)
Affinity screening 87 (1)
Soluble scFv production and purification 87 (4)
References 89 (2)
ARM complexes for in vitro display and 91 (20)
evolution of antibody combining sites
Michael J. Taussig
Maria A. T. Groves
Margit Menges
Hong Liu
Mingyue He
Introduction 91 (2)
Ribosome display methodology 93 (12)
Outline of procedure 93 (1)
Primer design and single chain antibody 93 (3)
(VH/K) construction for ARM display
Generation of ARM complexes by coupled 96 (2)
transcription/translation in vitro
Antigen selection of ARM complexes 98 (2)
Recovery and amplification of DNA from 100 (3)
antigen-selected ARM complexes
Further ARM cycles and cloning 103 (1)
Analysis of clones encoding antibodies by 104 (1)
ARM display
Examples of ARM display 105 (2)
ARM specificity 105 (1)
Selection of DB3R VH/K from libraries 106 (1)
Troubleshooting 107 (1)
Background 107 (1)
No DNA recovery 107 (1)
Summary 107 (4)
References 109 (2)
Human monoclonal antibodies to blood group 111 (14)
antigens
Belinda M. Kumpel
Introduction 111 (1)
General equipment and reagents required 112 (1)
Selection of donor 113 (1)
Preparation of lymphocytes 113 (1)
EBV transformation of B cells 114 (3)
Preparation of EBV 115 (1)
EBV transformation of B cells and growth 116 (1)
of B-LCL
Selection of antigen-specific B cells by 117 (1)
rosetting
Fusion of B-LCL with murine myeloma cells 118 (2)
(P3X63Ag8.653)
Screening techniques 120 (1)
Cloning 121 (1)
Cryopreservation of cells 122 (3)
References 123 (2)
Laboratory based methods for small scale 125 (24)
production of monoclonal antibodies
Bryan Griffiths
Introduction 125 (1)
General principles 125 (4)
Culture parameters 125 (2)
Medium and serum 127 (2)
Stationary cultures 129 (2)
Tissue culture plates and flasks 129 (1)
Specialized (scale-up) culture systems 130 (1)
Stirred cultures 131 (4)
Spinner flasks 131 (2)
SuperSpinner 133 (1)
Stirred bioreactors 134 (1)
Dynamic (non-stirred) culture systems 135 (2)
Roller bottle culture 135 (1)
Airlift fermenters 136 (1)
Perfusion (high cell density) systems 137 (6)
Hollow fibre (Acusyst) culture 138 (1)
Tecnomouse 138 (1)
Membrane culture systems (miniPERM) 139 (1)
Packed bed systems 140 (2)
Fluidized bed bioreactors 142 (1)
Harvesting and concentration 143 (2)
Harvesting and clarification 143 (1)
Concentration 144 (1)
Summary 145 (4)
References 146 (3)
Isolation and purification of monoclonal 149 (32)
antibodies from tissue culture supernatant
Geoff Hale
Objectives of antibody purification 149 (1)
Essential information about the antibody 150 (7)
Problems with purifying antibodies from 157
culture supernatant
Low concentration of antibody in culture 151 (1)
supernatant compared with serum
Potential contamination by bovine IgG 152 (1)
Equipment for antibody purification 153 (3)
Equipment for chromatography 154 (2)
Precipitation methods 156 (2)
Affinity chromatography 158 (4)
Reuse of affinity columns 159 (1)
Choice of affinity ligand 160 (2)
Immunoaffinity purification 162 (1)
Ion exchange chromatography 162 (4)
Cation exchange chromatography 163 (2)
Anion exchange chromatography 165 (1)
Immobilized metal affinity chromatography 166 (2)
(IMAC)
Size exclusion chromatography (SEC) 168 (1)
Hydrophobic interaction chromatography 169 (3)
Other chromatographic methods 172 (1)
Choice of method 173 (1)
Purification of antibody fragments 173 (1)
Storage of antibodies 173 (1)
Analysis of purity and activity 174 (5)
Antibody concentration and purity 174 (3)
Endotoxin contamination 177 (2)
Conclusion 179 (2)
Acknowledgements 179 (1)
References 180 (1)
Antibody production in plants 181 (26)
Pascal Drake
Eva Stoger
Liz Nicholson
Paul Christou
Julian K.-C. Ma
Introduction 181 (1)
Expressing recombinant proteins in plants 181 (5)
Plant hosts 182 (1)
Antibodies in plants 182 (3)
Modified viruses for transient expression 185 (1)
in plants
Glycosylation of recombinant proteins in 185 (1)
transgenic plants
Plant transformation 186 (10)
Gene constructs 187 (1)
Agrobacterium tumefaciens-mediated 188 (4)
transformation
Principles of particle bombardment 192 (4)
Plant transformation techniques 196 (5)
Agrobaderium-mediated transformation of 196 (3)
tobacco
Transformation of wheat by 199 (2)
micro-projectile bombardment
Screening regenerated plantlets for 201 (1)
immunoglobulin chain production
Self and cross-fertilization of 202 (1)
transgenic plants
The overall advantages in expressing 202 (5)
antibodies in plants
References 203 (4)
Radiolabelling of monoclonal antibodies 207 (30)
Stephen J. Mather
Introduction 207 (1)
The choice of radionuclide 207 (1)
In vitro applications of radiolabelled 207 (10)
antibodies
Iodine-125 208 (9)
In vivo applications of radiolabelled 217 (17)
antibodies
Iodine-123 218 (1)
Indium-111 219 (4)
Technetium-99m 223 (8)
Iodine-131 231 (1)
Yttrium-90 2 (233)
Other therapeutic radionuclides 233 (1)
Antibody fragments and genetically 234 (1)
engineered constructs
Quality control of radiolabelled antibodies 234 (3)
References 235 (2)
Non-radioactive antibody probes 237 (10)
G. Brian Wisdom
Introduction 237 (1)
Choice of label 238 (1)
General aspects of labelling 238 (2)
The labelling reactions 238 (1)
The monoclonal antibody 239 (1)
Scale and ratios 239 (1)
Purification and storage of the labelled 239 (1)
antibody
Labeiling with an enzyme 240 (2)
Labelling with fluorescein 242 (1)
Labelling with biotin 243 (1)
Labelling with digoxigenin (DIG) 244 (1)
Evaluation of labelled monoclonal antibodies 244 (3)
References 246 (1)
Immunogold probes for light and electron 247 (18)
microscopy
Paul Monaghan
David Robertson
Introduction 247 (2)
Pre-embedding labelling for SEM and TEM 249 (2)
Thawed cryosections 251 (4)
Progressive lowering of temperature 255 (1)
embedding (PLT)
R

Monoclonal Antibodies: A Practical Approach covers the preparation, testing, derivation, and applications of monoclonal antibodies. New immunological techniques incorporating tried and tested methodologies are described, making the book of interest to established and inexperiencedimmunologists. Both the standard somatic hybridization technique and recombinant techniques, including the use of phage libraries, for the preparation of rodent and human monoclonal antibodies are described. Protocols for both the small and large scale production are detailed, as well aspurification and labelling (with both radioisotopes and non-radioisotopes) methods. The applications of monoclonal antibodies in immunoblotting, enzyme linked immunoassays, immunofluorescence, and FACS analysis are all covered in detail. Finally protocols are given for the use of monoclonalantibodies in rheumatoid arthritis, tissue typing, detecting DNA modified during chemotherapy, and in the clinical analysis of transplantation samples for malignancy. This book will therefore be an invaluable laboratory companion to anyone using monoclonal antibodies in their research.