Saerens D., Muyldermans S. (Ed.) Single Domain Antibodies: Methods and Protocols

Humana Press, 2012. — 573 p.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.Overview of Single Domain Antibodies
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
Single Domain Antibody Library Construction
Creation of the Large and Highly Functional Synthetic Repertoire of Human VH and Vκ Domain Antibodies
Preparation of a Naïve Library of Camelid Single Domain Antibodies
Selection of 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 Aβ Depositions Using Complex Immune Libraries
Expression of Single Domain Antibodies and Derivatives
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 V: Improvement and Applications of Single Domain Antibodies
Improvement and Applications of Single Domain Antibodies
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 with an 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 Hishemistry
Case Studies
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