This volume covers current and emerging techniques for studying single-domain antibodies (sdAbs). Chapters guide readers through the biology and immunology of sdAbs in camelids and sharks, isolation of sdAbs, protein engineering approaches to optimize the solubility, stability, valency and antigen binding affinity of sdAbs, and specialized applications of sdAbs. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Authoritative and cutting-edge, Single-Domain Antibodies: Methods and Protocols aims to be a useful, practical guide to help researchers further their studies in this field.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt

Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in Camelids to a Wide Range of Useful Applications.- Overview, Generation, and Significance of Variable New Antigen Receptors (VNARs) as a Platform for Drug and Diagnostic Development.- Llama DNA Immunization and Isolation of Functional Single-Domain Antibody Binders.- Preparation of Immune and Synthetic VNAR Libraries as Sources of High-Affinity Binders.- Isolation of Single-Domain Antibodies to Transmembrane Proteins Using Magnetized Yeast Cell Targets.- A Transgenic Heavy Chain IgG Mouse Platform as a Source of High Affinity Fully Human Single-Domain Antibodies for Therapeutic Applications.- Cytoplasmic Production of Nanobodies and Nanobody-Based Reagents by Co-Expression of Sulfhydryl Oxidase and DsbC Isomerase.- Small Scale Secretory VHH Expression in Saccharomyces cerevisiae.- Production of Single-Domain Antibodies in Pichia pastoris.- Production of Designer VHH-Based Antibodies in Plant.- Assessing the Aggregation Propensity of Single-Domain Antibodies Upon Heat-Denaturation Employing the T m Shift.- Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing.- Engineering pH-Sensitive Single-Domain Antibodies.- Humanization of Camelid Single-Domain Antibodies.- Creation of Multimeric Single-Domain Antibodies Using Bacterial Superglues.- Introducing Cysteines into Nanobodies for Site-Specific Labelling.- Affinity-Guided Site Selective Labelling of Nanobodies With Aldehyde Handles.- Cytoplasmic Expression of Nanobodies with Formylglycine Generating Enzyme Tag and Conversion to a Bio-Orthogonal Aldehyde Group.- Site-Specific Fluorescent Labelling, Single-Step Immunocytochemistry, and Delivery of Nanobodies into Living Cells.- Design and Validation of Site-Specifically Labelled Single-Domain Antibody-Based Tracers for In Vivo Fluorescence Imaging and Image-Guided Surgery..- Design and Preparation of Photobodies: Light-Activated Single-Domain Antibody Fragments.- Visualizing Filoviral Nucleoproteins Using Nanobodies Fused to the Ascorbate Peroxidase Derivatives APEX2 and dEAPX.- Development of Glypican-2 Targeting Single-Domain Antibody CAR T Cells for Neuroblastoma.- Single-Domain Antibodies for Intracellular Toxin Neutralization.- Generation of Single-Domain Antibody-Based Recombinant Immunotoxins.- X-Ray Crystal Structure Analysis of VHH-Protein Antigen Complexes.- Functionalization of Magnetic Beads with Biotinylated Nanobodies for MALDI-TOF/MS-Based Quantitation of Small Analytes.- Nanobody-Based Assays for the Detection of Environmental and Agricultural Contaminants.- Peptide Tag-Specific Nanobodies for Studying Proteins in Live Cells.- Nanobody-Based GFP Traps to Study Protein Localization and Function in Developmental Biology.- Optogenetic Activation of Intracellular Nanobodies.