Antibodies are indispensable tools for research, diagnosis, and therapy. Recombinant approaches allow the modification and improvement of nearly all antibody properties, such as affinity, valency, specificity, stability, serum half-life, effector functions, and immunogenicity."Antibody Engineering" provides a comprehensive toolbox covering the well-established basics but also many exciting new techniques. The protocols reflect the latest "hands on" knowledge of key laboratories in this still fast-moving field. Newcomers will benefit from the proven step-by-step protocols, which include helpful practical advice; experienced antibody engineers will appreciate the new ideas and approaches. The book is an invaluable resource for all those engaged in antibody research and development.
Inhaltsverzeichnis
Cloning of the Antigen-binding Site from Hybridoma. - Cloning of Variable Domains from Mouse Hybridoma by PCR. - Coning Hybridoma cDNA by RACE. - Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly. - Generation of Antibody Repertoires. - Mouse Immune Libraries for the Generation of ScFv Fragments Directed Against Human Cell Surface Antigens. - Human Antibody Gene Libraries. - Synthetic Antibody Libraries. - Immune Libraries from Nonhuman Primates (NHP). - Generation of Rabbit Immune Libraries. - Selection of Antibody Fragments from Combinatorial Libraries. - Immunotube Selections. - Phage Display and Selection in Microtitre Plates. - Phage Display and Selections on Biotinylated Antigens. - Phage Display and Subtractive Selection on Cells. - Selection of Phage Antibody Libraries for Binding and Internalization into Mammalian Cells. - Improving Phage Display Throughput by Using Hyperphage, Miniaturized Titration and pVIII (g8p) ELISA. - Yeast Display and Selections. - The Generation of Transgenic Mice Expressing Human Antibody Repertoires. - Selection of Antibody Fragments by Means of the Filter-Sandwich Colony Screening Assay. - Semi-automated Magnetic Bead-Based Antibody Selection from Phage Display Libraries. - Engineering and Production of Immunoglobulins. - Aspects of Isotype Selection. - Generation of Heavy and Light Chains (Chimeric Antibodies). - Humanising Antibodies by CDR Grafting. - Humanization by Resurfacing. - Human Antibodies by Guided Selection. - In Silico De-Immunization. - Affinity Maturation by Chain Shuffling and Site Directed Mutagenesis. - Affinity Maturation by Random Mutagenesis and Phage Display. - Engineering of the Fc Region for Improved PK (FcRn Interaction). - Antibody-Dependent Enzyme Prodrug Therapy (ADEPT). - Production of Recombinant Human IgG Antibodies in the Baculovirus Expression System. - Expression of IgA Molecules in Mammalian Cells. - Antibody Characterization. - Expression of Complete Antibodies in Transgenic Plants. - Expression ofFull Length Monoclonal Antibodies (mAb) in Algal Chloroplast. - Expression of IgG Antibodies in Mammalian Cells. - Protein A/G Chromatography. - Epitope Analysis Using Synthetic Peptide Repertoires Prepared by SPOT Synthesis Technology. - Epitope Mapping by Printed Peptide Libraries. - Antibody Epitope Mapping Using Yeast Display. - Size Exclusion Chromatography. - Structural Characterization of Antibodies by Mass Spectrometry. - Antibody Glycans Characterization. - Affinity Measurements by Competition ELISA. - Anti-Histidine Antibodies as Tools for Reversible Capturing of His-Tagged Fusion Proteins for Subsequent Binding Analysis. - Affinity Measurements Using Quartz Crystal Microbalance (QCM). - Affinity Measurements with Radiolabeled Antibodies. - Neutralization Tests. - Functional Characterization of Antibodies Neutralizing Soluble Factors In Vitro and In Vivo. - Competitive ELISA. - Quantification of Human IgG and Related Fc Fusion Proteins by a Human IgG/Fc Capture ELISA. - Determination of Fc-Mediated Antibody-Effector Functions by Chromium Release Assay. - Binding Studies with Flow Cytometry.
