Generation of human single domain antibody repertoires by Kunkel mutagenesis

Romain Rouet; Kip Dudgeon; Daniel Christ

doi:10.1007/978-1-61779-974-7_10

Generation of human single domain antibody repertoires by Kunkel mutagenesis

Methods Mol Biol. 2012:907:195-209. doi: 10.1007/978-1-61779-974-7_10.

Authors

Romain Rouet¹, Kip Dudgeon, Daniel Christ

Affiliation

¹ Garvan Institute of Medical Research, Darlinghurst/Sydney, NSW, Australia.

PMID: 22907352
DOI: 10.1007/978-1-61779-974-7_10

Abstract

Human antibody single domains are a promising new class of antibody fragments. Here we describe methods for the cloning of human V(H) and V(L) genes into phage and phagemid vectors. Furthermore, we provide detailed protocols for the generation of single domain antibody libraries by Kunkel mutagenesis and the analysis of diversity by DNA sequencing and superantigen binding.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Surface Display Techniques
DNA / biosynthesis
Electrophoresis, Agar Gel
Electroporation
Enzyme-Linked Immunosorbent Assay
Escherichia coli / metabolism
Genetic Vectors / genetics
Humans
Immunoglobulin Heavy Chains / genetics
Immunoglobulin Light Chains / genetics
Molecular Biology / methods*
Mutagenesis / genetics*
Polymerase Chain Reaction
Sequence Analysis, DNA
Single-Domain Antibodies / genetics*
Templates, Genetic
Transformation, Genetic

Substances

Immunoglobulin Heavy Chains
Immunoglobulin Light Chains
Single-Domain Antibodies
DNA