Titlebook: Single-Domain Antibodies; Methods and Protocol Greg Hussack,Kevin A. Henry Book 2022 The Editor(s) (if applicable) and The Author(s), under

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Book 2022 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..

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Overview, Generation, and Significance of Variable New Antigen Receptors (VNARs) as a Platform for Dgether these unique properties position the VNAR platform as an alternative non-antibody binding domain for therapeutic drug, diagnostic and reagent development. In this introductory chapter, we highlight recent VNAR advancements that further underline the exciting potential of this discovery platform.

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Cytoplasmic Production of Nanobodies and Nanobody-Based Reagents by Co-Expression of Sulfhydryl Oxid independently of their redox requirements. Among other examples, the protocol described here was used to successfully produce nanobody fusions with fluorescent proteins that do not fold correctly in the periplasm, nanobodies with Fc domains, and nanobodies containing free cysteine tags.

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Production of Single-Domain Antibodies in e present methods for the cloning of sdAb-encoding genes into a . expression vector, production and purification of sdAbs, and measurement of sdAb-binding kinetics. Functional sdAbs are easily and routinely obtained using these methods.

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Nanobodies: From Serendipitous Discovery of Heavy Chain-Only Antibodies in Camelids to a Wide Range ated to possess multiple biochemical and biophysical advantages over other antigen-binding antibody fragments and alternative scaffolds. Today, nanobodies have a significant and growing impact on research, biotechnology, and medicine.

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Engineering pH-Sensitive Single-Domain Antibodiesof histidine substitution is sampled within the confines of the specified interface region(s). Finally, we describe phage display protocols for the selection and analysis of unique pH-dependent sdAb clones.

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Humanization of Camelid Single-Domain Antibodiesn and complementarity-determining region grafting strategies to empirical scoring for prioritization of back-mutations, step-by-step instructions, and templates are provided along with bioinformatics resources to assist each step of the humanization process. Alternative approaches, warnings, and caveats are also presented.

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