Interest in recombinant antibody technologies has rapidly increased because of the wide range of possible applications in therapy and diagnosis, especially in cancer treatment. The possibility of generating human antibodies that are not accessible by conventional polyclonal or monoclonal approaches has forced the development of antibody engineering technologies even more.
This manual presents a comprehensive collection of detailed, step-by-step protocols provided by experts in the field. All basic methods needed in antibody engineering - not only methods to generate recombinant antibodies, but also protocols for analysis and their use - and recently developed and emerging technologies are covered. In particular, protocols on the following topics are provided:
Hybridoma immortalisation Generation and screening of antibody gene libraries from human donors, mice and rabbits Antibody selection on immunotubes, cells, tissues; proximity and step-back selections Creation of human monoclonal antibodies to toxic or highly pathogenic agents without immunisation Improvment of antibody binding Antibody humanisation Genetic fusions for the production of multifunctional antibody derivatives Radiolabelled recombinant antibodies Bispecific antibodies Antibody - enzyme fusions Intracellular antibodies Determination of affinity and specificity Computer analysis of antibody sequence and structure Epitope analysis by various phage display systems and peptide spot membranes Eukaryotic (plant, baculovirus, yeast, mammalian cells) and prokaryotic production systems for recombinant antibodies Purification systems Xenograft mice Emerging technologies

Inhalt

1 Recombinant Antibodies.- Generation of Recombinant Antibodies from Hybridoma.- 2 Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly.- 3 Construction of scFv from Hybridoma by Two-Step Cloning.- 4 Cloning Hybridoma cDNA by RACE.- Generation of Antibody Libraries.- 5 Phage-Display Libraries of Murine Antibody Fab Fragments.- 6 Generation of Naive Human Antibody Libraries.- 7 Generation of Antibody Gene Libraries from Seropositive Human Donors.- 8 Generation of Rabbit Immune Libraries.- Isolation of Antibody Fragments from Combinatorial Libraries.- 9 Immunotube Selections.- 10 Selections on Biotinylated Antigens.- 11 Guided Selections on Cells.- 12 Proximity (ProxiMol) and Step-Back Selections.- 13 Selections on Tissue Sections.- 14 Selections of Human Antibody Fragments by Anti-Idiotypic Interaction.- 15 Selecting Intracellular Antibodies Using the Two-Hybrid System.- 16 Selection of Phage Antibody Libraries for Binding and Internalization Into Mammalian Cells.- Expression and Purification of Antibody Fragments in Bacteria.- 17 Purification and Characterisation of His-Tagged Antibody Fragments.- 18 Functional Minipreps of scFv-Antibody Fragments.- 19 Expression of scFv in L-Form Bacteria.- 20 Expression and Purification of Fab Fragments.- 21 Purification and Analysis of Antibody Fragments Using Proteins L, A and LA.- 22 Purification and Analysis of Strep-Tagged Antibody Fragments.- 23 Improving Expression of scFv Fragments by Coexpression of Periplasmic Chaperons.- Expression and Purification of Antibody Fragments in Eukaryotic Cells.- 24 Expresssion of scFv Antibody Fragments in the Yeast Pichia Pastoris.- 25 Production of Recombinant Human IgG Antibodies in the Baculovirus Expresssion System.- 26 Expresssion of Antibodies in Mammalian Cells.- 27 Expresssion of Antibody Fragments in Plant Cells.- Determination of Affinities.- 28 Affinity Measurements by Band Shift and Competition ELISA.- 29 Affinity Measurements of Antibody Fragments on Phage by Quartz Crystal Microbalance (QCM).- Sequence and Structure Analysis and Modelling.- 30 Sequence Analysis of Human Antibody Genes.- 31 Protein Sequence and Structure Analysis of Antibody Variable Domains.- Epitope Mapping.- 32 Epitope Mapping with Synthetic Peptides Prepared by SPOT Synthesis.- 33 Epitope Mapping with Random Peptide Libraries.- 34 Epitope Mapping with Gene Fragment Libraries.- In vivo Experiments.- 35 Imaging Tumor Xenografts Using Radiolabeled Antibody Fragments.- 36 Xenograft Mouse Models for Tumour Targeting.- Affinity Maturation of Antibody Fragments.- 37 Affinity Maturation by Chain Shuffling and Site Directed Mutagenesis.- 38 Affinity and Specificity Maturation by CDR Walking.- Humanisation of Antibody Fragments.- 39 Humanisation by Guided Selection.- 40 Humanising Antibodies by CDR Grafting.- Antibody Engineering to Improve Stability.- 41 Stabilization Strategies and Application of recombinant Fvs and Fv Fusion Proteins.- Bivalent and Bispecific Antibody Fragments.- 42 Bivalent and Bispecific Diabodies and Single-Chain Diabodies.- 43 Miniantibodies.- 44 ScFv-CH3 and scFv-Fc Fusion Proteins.- 45 CTL-Assays for Functional Testing of Bispecific Antibody Fragments.- Recombinant Antibody-Fusion Proteins.- 46 Antibody-Enzyme Fusions.- 47 Cytokine-scFv Fusion Proteins.- 48 Antibody Fusion Proteins for Targeted Gene Delivery.- Intracellular Targeting of Antibody Fragments.- 49 Intracellular Single Chain Antibodies - Methods for Derivation and Employment.- 50 Expresssing Intracellular Single-Chain Fv Fragments in Mammalian Cells.- Appendices.- Appendix 1 Internet Resources.- Appendix 2 Amino Acids: Nomenclature and Codons.