Antibody Engineering Volume 1 (eBook)

Foreword 5
Preface 6
Contents 7
Cloning of the Antigen-binding Site from Hybridoma 12
Cloning of Variable Domains from Mouse Hybridoma by PCR 13
Coning Hybridoma cDNA by RACE 25
Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly 31
Generation of Antibody Repertoires 55
Mouse Immune Libraries for the Generation of ScFv Fragments Directed Against Human Cell Surface Antigens 56
Human Antibody Gene Libraries 73
Synthetic Antibody Libraries 93
Immune Libraries from Nonhuman Primates ( NHP) 106
Generation of Rabbit Immune Libraries 122
Selection of Antibody Fragments from Combinatorial Libraries 131
Immunotube Selections 132
Phage Display and Selection in Microtitre Plates 143
Phage Display and Selections on Biotinylated Antigens 154
Phage Display and Subtractive Selection on Cells 168
Selection of Phage Antibody Libraries for Binding and Internalization into Mammalian Cells 186
Improving Phage Display Throughput by Using Hyperphage, Miniaturized Titration and pVIII ( g8p) ELISA 199
Yeast Display and Selections 209
The Generation of Transgenic Mice Expressing Human Antibody Repertoires 236
Selection of Antibody Fragments by Means of the Filter- Sandwich Colony Screening Assay 256
Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries 268
Engineering and Production of Immunoglobulins 289
Aspects of Isotype Selection 290
Generation of Heavy and Light Chains ( Chimeric Antibodies) 306
Humanising Antibodies by CDR Grafting 317
Humanization by Resurfacing 338
Human Antibodies by Guided Selection 351
In Silico De-Immunization 365
Affinity Maturation by Chain Shuffling and Site Directed Mutagenesis 373
Affinity Maturation by Random Mutagenesis and Phage Display 393
Engineering of the Fc Region for Improved PK ( FcRn Interaction) 406
Antibody-Dependent Enzyme Prodrug Therapy ( ADEPT) 426
Production of Recombinant Human IgG Antibodies in the Baculovirus Expression System 447
Expression of IgA Molecules in Mammalian Cells 465
Antibody Characterization 481
Expression of Complete Antibodies in Transgenic Plants 482
Expression of Full Length Monoclonal Antibodies ( mAb) in Algal Chloroplast 496
Expression of IgG Antibodies in Mammalian Cells 510
Protein A/G Chromatography 523
Epitope Analysis Using Synthetic Peptide Repertoires Prepared by SPOT Synthesis Technology 528
Epitope Mapping by Printed Peptide Libraries 563
Antibody Epitope Mapping Using Yeast Display 580
Size Exclusion Chromatography 595
Structural Characterization of Antibodies by Mass Spectrometry 601
Antibody Glycans Characterization 623
Affinity Measurements by Competition ELISA 645
Anti-Histidine Antibodies as Tools for Reversible Capturing of His- Tagged Fusion Proteins for Subsequent Binding Analysis 654
Affinity Measurements Using Quartz Crystal Microbalance ( QCM) 669
Affinity Measurements with Radiolabeled Antibodies 680
Neutralization Tests 690
Functional Characterization of Antibodies Neutralizing Soluble Factors In Vitro and In Vivo 708
Competitive ELISA 723
Quantification of Human IgG and Related Fc Fusion Proteins by a Human IgG/ Fc Capture ELISA 727
Determination of Fc-Mediated Antibody- Effector Functions by Chromium Release Assay 733
Binding Studies with Flow Cytometry 748
Index 763