Single Domain Antibodies

This Methods in Molecular Biology(TM) book covers single variable domain antibody fragments, which are the smallest intact antigen-binding fragments that can be produced recombinantly at low cost. Includes materials lists, protocols and troubleshooting tips.

The development of the hybridoma technology created the possibility to obtain unlimited amounts of monoclonal antibodies (mAb) with high specificity and affinity for any target and to introduce mAbs in a wide range of applications; however, the bulky size of mAbs, costly production, and cumbersome engineering hampered regularly their streamlined development in some applications. In Single Domain Antibodies: Methods and Protocols, expert researchers examine single variable domain antibody fragments, referred to as VH, VL, VHH or VNAR. These fragments are the smallest intact antigen-binding fragments that can be produced recombinantly at low cost. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Features methods of selection, expression, improvement, and library construction of single domain antibodies Includes step-by-step instruction that has been tested in labs around the world Provides key tips and implementation advice that is essential for success Includes supplementary material: sn.pub/extras

Inhalt

From Whole Monoclonal Antibodies to Single Domain Antibodies: Think Small.- Introduction to Heavy Chain Antibodies and Derived Nanobodies.- Overview and Discovery of IgNARs and Generation of VNARs.- Creation of the Large and Highly Functional Synthetic Repertoire of Human VH and V Domain Antibodies.- Preparation of a Naïve Library of Camelid Single Domain Antibodies.- Selection by Phage Display of Single Domain Antibodies Specific to Antigens in Their Native Conformation.- Semi-Automated Panning of Naive Camelidae Libraries and Selection of Single-Domain Antibodies Against Peptide Antigens.- Pichia Surface Display: A Tool for Screening Single Domain Antibodies.- Bacterial Two Hybrid: A Versatile One-Step Intracellular Selection Method.- Intracellular Antibody Capture (IAC) Methods for Single Domain Antibodies.- Selection of Functional Single Domain Antibody Fragments for Interfering with Protein-Protein Interactions Inside Cells: A One Plasmid Mammalian Two-Hybrid System.- Cell-Free Selection of Domain Antibodies by In Vitro Compartmentalization.- Selection of VHHs Under Application Conditions.- Isolation and Characterization of Clostridium difficile Toxin-Specific Single-Domain Antibodies.- Selection of VHH Antibody Fragments that Recognize Different A Depositions Using Complex Immune Libraries.- Expression of Single-Domain Antibodies in Bacterial Systems.- Expression of VHHs in S. cerevisiae.- Stable Expression of Chimeric Heavy Chain Antibodies in CHO Cells.- Production of Camel-Like Antibodies in Plants.- Selecting and Purifying Autonomous Human Variable Heavy (VH) Domains.- Solubility and Stability Engineering of Human VH Domains.- Improvement of Proteolytic Stability through In Silico Engineering.- Selection of Human VH Single Domains with Improved Biophysical Properties by Phage Display.- Improvement of Single Domain Antibody Stability by Disulfide Bond Introduction.- Characterization of Single-Domain Antibodies withan Engineered Disulfide Bond.- Affinity Maturation of Single-Domain Antibodies by Yeast Surface Display.- Multivalent Display of Single-Domain Antibodies.- Methods for Determining the PK Parameters of AlbudAbs and of Long Serum Half Life Drugs Made Using the AlbudAb Technology.- Fluorescent Protein Specific Nanotraps to Study Protein-Protein Interactions and Histone-Tail Peptide Binding.- Site-Specific Labeling of His-Tagged Nanobodies with 99mTc: A Practical Guide.- Nanobody-Based Chromatin Immunoprecipitation.- User-Friendly Expression Plasmids Enable the Fusion of VHHs to Application-Specific Tags.- Application of Single Domain Antibodies in Tumor Histochemistry.- Nanobodies as Structural Probes of Protein Misfolding and Fibril Formation.- Molecular Imaging Using Nanobodies: A Case Study.- Case Study on Live Cell Apoptosis-Assay Using Lamin-Chromobody Cell-Lines for High-Content Analysis.