Generation of human single domain antibody repertoires by Kunkel mutagenesis.
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.
Garvan Institute of Medical Research, Darlinghurst/Sydney, NSW, Australia.
Romain
R
Rouet
Kip
K
Dudgeon
Daniel
D
Christ
D061505:Cell Surface Display Techniques; D004247:DNA / Q000096:biosynthesis; D004587:Electrophoresis, Agar Gel; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D016296:Mutagenesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D061905:Single-Domain Antibodies / Q000235:genetics*; D013698:Templates, Genetic; D014170:Transformation, Genetic
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D061905:Single-Domain Antibodies; D004247:DNA