Abstract:

This comprehensive collection of established antibody phage display protocols features authoritative guidance that will enable the nonspecialist successfully to carry them out. Coverage spans the construction of antibody libraries, the selection of antibody clones with the desired properties, and their modification, expression, and purification. Comprehensive and highly practical, Antibody Phage Display: Methods and Protocols provides biochemists, molecular biologists, and immunologists with a gold-standard reference guide to the successful isolation, modification, and expression of recombinant antibodies using today's powerful phage display technology.

Preface v
Contributors xi
1 Overview of Antibody Phage-Display Technology and Its Applications 1
2 Standard Protocols for the Construction of Fab Libraries 39
3 Standard Protocols for the Construction of scFv Libraries 59
4 Broadening the Impact of Antibody Phage Display Technology: Amplification of Immunoglobulin Sequences from Species Other than Humans or Mice 73
5 Construction of Large Naive Fab Libraries 87
6 Construction of Polyclonal Antibody Libraries Using Phage Display 101
7 Antigen-Driven Stimulation of B-Lymphocytes In Vitro 113
8 The Recovery of Immunoglobulin Sequences from Single Human B Cells by Clonal Expansion 121
9 Panning of Antibody Phage-Display Libraries: Standard Protocols 133
10 Selection of Antibodies Against Biotinylated Antigens 147
11 Isolation of Anti-Hapten Specific Antibody Fragments from Combinatorial Libraries 159
12 Blocking Immunodominant Epitopes by Competitive Deselection 173
13 Rescue of a Broader Range of Antibody Specificities Using an Epitope-Masking Strategy 179
14 Screening of Phage-Expressed Antibody Libraries by Capture Lift 187
15 Antibody-Guided Selection Using Capture-Sandwich ELISA 195
16 Proximity-Guided (ProxiMol) Antibody Selection 201
17 Isolation of Human Monoclonal Antibodies Using Guided Selection with Mouse Monoclonal Antibodies 207
18 Selecting Antibodies to Cell-Surface Antigens Using Magnetic Sorting Techniques 219
19 Isolation of Human Tumor-Associated Cell Surface Antigen-Binding scFvs 227
20 Subtractive Isolation of Single-Chain Antibodies Using Tissue Fragments 235
21 Selection of Antibodies Based on Antibody Kinetic Binding Properties 245
22 Selection of Functional Antibodies on the Basis of Valency 255
23 Two-Step Strategy for Alteration of Immunoglobulin Specificity by In Vitro Mutagenesis 259
24 Targeting Random Mutations to Hotspots in Antibody Variable Domains for Affinity Improvement 269
25 Error-Prone Polymerase Chain Reaction for Modification of scFvs 287
26 Use of Escherichia coli Mutator Cells to Mature Antibodies 295
27 Chain Shuffling to Modify Properties of Recombinant Immunoglobulins 303
28 Generation of Bispecific and Tandem Diabodies 317
29 High-Level Periplasmic Expression and Purification of scFvs 333
30 Periplasmic Expression and Purification of Recombinant Fabs 343
31 Expression of Antibody Fragments in Pichia pastoris 349
32 Expression of V[subscript HH] Antibody Fragments in Saccharomyces cerevisiae 359
33 Intrabodies: Targeting scFv Expression to Eukaryotic Intracellular Compartments 367
34 Expression of scFvs and scFv Fusion Proteins in Eukaryotic Cells 379
35 Expression of Antibody Fab Fragments and Whole Immunoglobulin in Mammalian Cells 389
Index 397